Supercomplex-associated Cox26 protein binds to cytochrome c oxidase.

Here we identified a hydrophobic 6.4kDa protein, Cox26, as a novel component of yeast mitochondrial supercomplex comprising respiratory complexes III and IV. Multi-dimensional native and denaturing electrophoretic techniques were used to identify proteins interacting with Cox26. The majority of the Cox26 protein was found non-covalently bound to the complex IV moiety of the III-IV supercomplexes. A population of Cox26 was observed to exist in a disulfide bond partnership with the Cox2 subunit of complex IV. No pronounced growth phenotype for Cox26 deficiency was observed, indicating that Cox26 may not play a critical role in the COX enzymology, and we speculate that Cox26 may serve to regulate or support the Cox2 protein. Respiratory supercomplexes are assembled in the absence of the Cox26 protein, however their pattern slightly differs to the wild type III-IV supercomplex appearance. The catalytic activities of complexes III and IV were observed to be normal and respiration was comparable to wild type as long as cells were cultivated under normal growth conditions. Stress conditions, such as elevated temperatures resulted in mild decrease of respiration in non-fermentative media when the Cox26 protein was absent.

LGR4 and LGR5 are R-spondin receptors mediating Wnt/ß-catenin and Wnt/PCP signalling.

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/ß-catenin and Wnt/PCP signalling. R-spondin-triggered ß-catenin signalling requires Clathrin, while Wnt3a-mediated ß-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.

DEAD box RNA helicases act as nucleotide exchange factors for casein kinase 2.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.

A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

Casein kinase 1 and Wnt/ß-catenin signaling.

Casein kinase 1 (CK1) members play a critical and evolutionary conserved role in Wnt/ß-catenin signaling. They phosphorylate several pathway components and exert a dual function, acting as both Wnt activators and Wnt inhibitors. Recent discoveries suggest that CK1 members act in a coordinated manner to regulate early responses to Wnt and notably that their enzymatic activity is regulated. Here, I provide a brief update of CK1 function and regulation in Wnt/ß-catenin signaling.

Secreted and transmembrane wnt inhibitors and activators.

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand-receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-ß and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease.

RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-ß-catenin signaling.

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-ß-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-ß-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

Transmembrane protein 198 promotes LRP6 phosphorylation and Wnt signaling activation.

Wnt/ß-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.

Polymorphisms in CTNNBL1 in relation to colorectal cancer with evolutionary implications.

Colorectal cancer (CRC) is a complex disease related to environmental and genetic risk factors. Several studies have shown that susceptibility to complex diseases can be mediated by ancestral alleles. Using RNAi screening, CTNNBL1 was identified as a putative regulator of the Wnt signaling pathway, which plays a key role in colorectal carcinogenesis. Recently, single nucleotide polymorphisms (SNPs) in CTNNBL1 have been associated with obesity, a known risk factor for CRC. We investigated whether genetic variation in CTNNBL1 affects susceptibility to CRC and tested for signals of recent selection. We applied a tagging SNP approach that cover all known common variation in CTNNBL1 (allele frequency >5%; r(2)>0.8). A case-control study was carried out using two well-characterized study populations: a hospital-based Czech population composed of 751 sporadic cases and 755 controls and a family/early onset-based German population (697 cases and 644 controls). Genotyping was performed using allele specific PCR based TaqMan® assays (Applied Biosystems, Weiterstadt, Germany). In the Czech cohort, containing sporadic cases, the ancestral alleles of three SNPs showed evidence of association with CRC: rs2344481 (OR 1.44, 95%CI 1.06-1.95, dominant model), rs2281148 (OR 0.59, 95%CI 0.36-0.96, dominant model) and rs2235460 (OR 1.38, 95%CI 1.01-1.89, AA vs. GG). The associations were less prominent in the family/early onset-based German cohort. Data derived from several databases and statistical tests consistently pointed to a likely shaping of CTNNBL1 by positive selection. Further studies are needed to identify the actual function of CTNNBL1 and to validate the association results in other populations.

Requirement of prorenin receptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling.

Wnt/beta-catenin signaling is important in stem cell biology, embryonic development, and disease, including cancer. However, the mechanism of Wnt signal transmission, notably how the receptors are activated, remains incompletely understood. We found that the prorenin receptor (PRR) is a component of the Wnt receptor complex. PRR functions in a renin-independent manner as an adaptor between Wnt receptors and the vacuolar H+-adenosine triphosphatase (V-ATPase) complex. Moreover, PRR and V-ATPase were required to mediate Wnt signaling during antero-posterior patterning of Xenopus early central nervous system development. The results reveal an unsuspected role for the prorenin receptor, V-ATPase activity, and acidification during Wnt/beta-catenin signaling.

Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.

Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling.

Kremen 1 and 2 (Krm1/2) are transmembrane receptors for Wnt antagonists of the Dickkopf (Dkk) family and function by inhibiting the Wnt co-receptors LRP5/6. Here we show that Krm2 functions independently from Dkks during neural crest (NC) induction in Xenopus. Krm2 is co-expressed with, and regulated by, canonical Wnts. Krm2 is differentially expressed in the NC, and morpholino-mediated Krm2 knockdown inhibits NC induction, which is mimicked by LRP6 depletion. Conversely, krm2 overexpression induces ectopic NC. Kremens bind to LRP6, promote its cell-surface localization and stimulate LRP6 signaling. Furthermore, Krm2 knockdown specifically reduces LRP6 protein levels in NC explants. The results indicate that in the absence of Dkks, Kremens activate Wnt/beta-catenin signaling through LRP6.

The MRH protein Erlectin is a member of the endoplasmic reticulum synexpression group and functions in N-glycan recognition.

Kremen1 and 2 (Krm1/2) are coreceptors for Dickkopf1 (Dkk1), an antagonist of Wnt/beta-catenin signaling, and play a role in head induction during early Xenopus development. In a proteomic approach we identified Erlectin, a novel protein that interacts with Krm2. Erlectin (XTP3-B) is member of a protein family containing mannose 6-phosphate receptor homology (MRH-, or PRKCSH-) domains implicated in N-glycan binding. Like other members of the MRH family, Erlectin is a luminal resident protein of the endoplasmic reticulum. It contains two MRH domains, of which one is essential for Krm2 binding, and this interaction is abolished by Krm2 deglycosylation. The overexpression of Erlectin inhibits transport of Krm2 to the cell surface. Analysis of its embryonic expression pattern in Xenopus reveals that Erlectin is member of the endoplasmic reticulum synexpression group. Erlectin morpholino antisense injection leads to head and axial defects during organogenesis stages in Xenopus embryos. The results indicate that Erlectin functions in N-glycan recognition in the endoplasmic reticulum, suggesting that it may regulate glycoprotein traffic.

Functional organization of the yeast proteome by systematic analysis of protein complexes.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.