Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling,is sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.     

New insight into the functions of the interleukin-17 receptor adaptor protein Act1 in psoriatic arthritis

Recent genome-wide association studies have implicated the tumor necrosis factor receptor-associated factor 3-interacting protein 2 (TRAF3IP2) gene and its product, nuclear factor-kappa-B activator 1 (Act1), in the development of psoriatic arthritis (PsA). The high level of sequence homology of the TRAF3IP2 (Act1) gene across the animal kingdom and the presence of the Act1 protein in multiple cell types strongly suggest that the protein is of importance in normal cellular function. Act1 is an adaptor protein for the interleukin-17 (IL-17) receptor, and recent observations have highlighted the significance of IL-17 signaling and localized inflammation in autoimmune diseases. This review summarizes data from recent genome-wide association studies as well as immunological and molecular investigations of Act1. Together, these studies provide new insight into the role of IL-17 signaling in PsA. It is well established that IL-17 activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling pathways normally leads to nuclear factor-kappa-B-mediated inflammation. However, the dominant PsA-associated TRAF3IP2 (Act1) gene single-nucleotide polymorphism (rs33980500) results in decreased binding of Act1 to TRAF6. This key mutation in Act1 could lead to a greater association of the IL-17 receptor with TRAF2/TRAF5 and this in turn suggests an alternative function for IL-17 in PsA. The recent observations described and discussed in this review raise the clinically significant possibility of redefining the immunological role of IL-17 in PsA and provide a basis for defining future studies to elucidate the molecular and cellular functions of Act1.     

The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling

TNFR-associated death domain protein (TRADD) is a key effector protein of TNFR1 signaling. However, the role of TRADD in other death receptor (DR) signaling pathways, including DR3, has not been completely characterized. Previous studies using overexpression systems suggested that TRADD is recruited to the DR3 complex in response to the DR3 ligand, TNF-like ligand 1A (TL1A), indicating a possible role in DR3 signaling. Using T cells from TRADD knockout mice, we demonstrate in this study that the response of both CD4(+) and CD8(+) T cells to TL1A is dependent upon the presence of TRADD. TRADD knockout T cells therefore lack the appropriate proliferative response to TL1A. Moreover, in the absence of TRADD, both the stimulation of MAPK signaling and activation of NF-κB in response to TL1A are dramatically reduced. Unsurprisingly, TRADD is required for recruitment of receptor interacting protein 1 and TNFR-associated factor 2 to the DR3 signaling complex and for the ubiquitination of receptor interacting protein 1. Thus, our findings definitively establish an essential role of TRADD in DR3 signaling.     

FGFR-ERK signaling is an essential component of tissue separation

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.     

Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling

Interleukin (IL)-1 plays an important role in inflammation and regulation of immune responses. The activated IL-1 receptor complex, which consists of the IL-1 receptor type I and the IL-1 receptor accessory protein (IL-1RAcP), generates multiple cellular responses including NF-kappaB activation, IL-2 secretion, and IL-2 promoter activation. Reconstitution experiments in EL4D6/76 cells lacking IL-1RAcP expression and IL-1 responsiveness were used to analyze structure-function relationships of the IL-1RAcP cytoplasmic tail. Mutating a potential tyrosine kinase phosphorylation motif and various conserved amino acid (aa) residues had no effect on IL-1 responsiveness. Truncation analyses revealed that box 3 of the TIR domain was required for NF-kappaB activation, IL-2 production, and c-Jun N-terminal kinase (JNK) activation, whereas IL-2 promoter activation was only partially inhibited. Surprisingly, deletion of aa 527-534 resulted in almost complete loss of all IL-1 responsiveness. Replacement of these aa with alanyl residues did not reconstitute NF-kappaB activation, IL-2 production, or JNK activation but partly restored IL-2 promoter activation. Immunoprecipitation data revealed a strong correlation between MyD88 binding with NF-kappaB activation and IL-2 production but not with IL-2 promoter activation. Taken together, our data indicate that box 3 of IL-1RAcP is critical for IL-1-dependent NF-kappaB activation and stabilization of IL-2 mRNA via JNK, whereas aa 527-534 largely contribute to IL-2 promoter activation.     

The Src-like adaptor protein 2 regulates colony-stimulating factor-1 receptor signaling and down-regulation

Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling.     

The LIM adaptor protein LMO4 is an essential regulator of neural crest development

The neural crest (NC) is a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates and migrate extensively before giving rise to diverse derivatives. A number of components of the neural crest gene regulatory network (NC-GRN) are used reiteratively to control multiple steps in the development of these cells. It is therefore important to understand the mechanisms that control the distinct function of reiteratively used factors in different cellular contexts, and an important strategy for doing so is to identify and characterize the regulatory factors they interact with. Here we report that the LIM adaptor protein, LMO4, is a Slug/Snail interacting protein that is essential for NC development. LMO4 is expressed in NC forming regions of the embryo, as well as in the central nervous system and the cranial placodes. LMO4 is necessary for normal NC development as morpholino-mediated knockdown of this factor leads to loss of NC precursor formation at the neural plate border. Misexpression of LMO4 leads to ectopic expression of some neural crest markers, but a reduction in the expression of others. LMO4 binds directly to Slug and Snail, but not to other components of the NC-GRN and can modulate Slug-mediated neural crest induction, suggesting a mechanistic link between these factors. Together these findings implicate LMO4 as a critical component of the NC-GRN and shed new light on the control of Snail family repressors.     

EpsinR is an adaptor for the SNARE protein Vti1b

EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1–depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a ~50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1–depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.     

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. We have identified loss-of-function mutations in dshc, the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain completely abolishes DSHC function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, DSHC is involved in signaling by only a subset of RTKs: dshc mutants show defects in Torso and DER but not Sevenless signaling, which is confirmed by epistasis experiments. We show by double-mutant analysis that the adaptors DOS, DRK, and DSHC act in parallel to transduce the Torso signal. Our results suggest that DSHC confers specificity to receptor signaling.     

The PYRIN-CARD protein ASC is an activating adaptor for caspase-1

The PYRIN and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the apoptotic and inflammatory signaling pathways. Here we show that the PYRIN-CARD protein ASC functions as a caspase-1-activating adaptor. ASC interacted specifically with procaspase-1 via CARD-CARD interactions and induced its oligomerization. Consistent with these results ectopic expression of full-length ASC, but not its isolated CARD or PYRIN domain, with procaspase-1 induced activation of procaspase-1 and processing of pro-interleukin-1beta in transfected cells. Substitution of the PYRIN domain of ASC with an inducible FKBP12 oligomerization domain produced a molecule that can induce caspase-1 activation in response to stimulation with the oligomerization drug AP20187, suggesting that the PYRIN domain functions as an oligomerization domain, whereas the CARD domain functions as the effector domain in the caspase-1 activation pathway. Furthermore stable expression of an isolated CARD of ASC in THP-1 cells diminished interleukin-1beta generation in response to pro-inflammatory cytokines. These results indicate that ASC is involved in the caspase-1 signaling pathway by mediating the assembly of a caspase-1-inflammasome signaling complex in response to pro-inflammatory cytokine stimulation.     

Interleukin-17F signaling requires ubiquitination of interleukin-17 receptor via TRAF6

Interleukin-17F (IL-17F), together with interleukin-17A (IL-17 or IL-17A), is a marker of T(H)17 cells, a new lineage of effector CD4(+) T cells to contribute to pathogenesis of a growing list of autoimmune and inflammatory diseases, such as experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA). IL-17F, similar to IL-17A, was reported to employ interleukin-17 receptor (IL-17R or IL-17RA) for signaling but the downstream cascades remain largely elusive. Here we report that TRAF6 interacts with IL-17R and mediates ubiquitination of the receptor. We observed that IL-17F and IL-17A could induce IL-17R ubiquitination and DN-TRAF6, a dominant-negative mutant, could block IL-17F- but not IL-17A-triggered ubiquitination of IL-17R. Moreover, we showed that the ubiquitination of IL-17R was positively correlated with the downstream signaling, as evaluated by a luciferase reporter driven by a putative native promoter of 24p3, an IL-17 targeted gene. Our results indicate that ubiquitination of IL-17R mediated by TRAF6 plays a critical role in IL-17F signaling. This study, for the first time, reveals a possible molecular mechanism that the initiation of the IL-17F/IL-17R signaling pathway requires the receptor ubiquitination by TRAF6.     

Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex.     

Rab27a is an essential component of melanosome receptor for myosin Va

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution in dilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5'-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.     

Synapse formation is regulated by the signaling adaptor GIT1

Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.