The NADPH oxidases DUOX1 and DUOX2 are sorted to the apical plasma membrane in epithelial cells via their respective maturation factors DUOXA1 and DUOXA2.

The membrane-integrated NADPH oxidases DUOX1 and DUOX2 are recruited to the apical plasma membrane in epithelial cells to release hydrogen peroxide, thereby playing crucial roles in various functions such as thyroid hormone synthesis and host defense. However, it has remained unknown about the molecular mechanism for apical sorting of DUOX1 and DUOX2. Here we show that DUOX1 and DUOX2 are correctly sorted to the apical membrane via the membrane-spanning DUOX maturation proteins DUOXA1 and DUOXA2, respectively, when co-expressed in MDCK epithelial cells.

TMEM25 is a Par3-binding protein that attenuates claudin assembly during tight junction development.

The tight junction (TJ) in epithelial cells is formed by integral membrane proteins and cytoplasmic scaffolding proteins. The former contains the claudin family proteins with four transmembrane segments, while the latter includes Par3, a PDZ domain-containing adaptor that organizes TJ formation. Here we show the single membrane-spanning protein TMEM25 localizes to TJs in epithelial cells and binds to Par3 via a PDZ-mediated interaction with its C-terminal cytoplasmic tail.

Hypoxia stabilizes the H O -producing oxidase Nox4 in cardiomyocytes via suppressing autophagy-related lysosomal degradation.

The hydrogen peroxide (H O )-producing NADPH oxidase Nox4, forming a heterodimer with p22 , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H O production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level.

GPR125 (ADGRA3) is an autocleavable adhesion GPCR that traffics with Dlg1 to the basolateral membrane and regulates epithelial apicobasal polarity.

The adhesion family of G protein-coupled receptors (GPCRs) is defined by an N-terminal large extracellular region that contains various adhesion-related domains and a highly-conserved GPCR-autoproteolysis-inducing (GAIN) domain, the latter of which is located immediately before a canonical seven-transmembrane domain. These receptors are expressed widely and involved in various functions including development, angiogenesis, synapse formation, and tumorigenesis. GPR125 (ADGRA3), an orphan adhesion GPCR, has been shown to modulate planar cell polarity in gastrulating zebrafish, but its biochemical properties and role in mammalian cells have remained largely unknown.

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts.

Intramolecular interaction in LGN, an adaptor protein that regulates mitotic spindle orientation.

Proper mitotic spindle orientation requires that astral microtubules are connected to the cell cortex by the microtubule-binding protein NuMA, which is recruited from the cytoplasm. Cortical recruitment of NuMA is at least partially mediated via direct binding to the adaptor protein LGN. LGN normally adopts a closed conformation via an intramolecular interaction between its N-terminal NuMA-binding domain and its C-terminal region that contains four GoLoco (GL) motifs, each capable of binding to the membrane-anchored Gα subunit of heterotrimeric G protein.

Differential cell surface recruitment of the superoxide-producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1.

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII-coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide-producing NADPH oxidase, is transported to the cell surface in a manner resistant to co-expression of Sar1 (H79G), a GTP-fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER-to-Golgi transport of glycoproteins including the Nox5-related oxidase Nox2.

Tropomyosin-related kinase B mediated signaling contributes to the induction of malignant phenotype of gallbladder cancer.

This study aims to demonstrate the clinical and biological significance of Brain derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) signaling in gallbladder cancer (GBC) through a series of in vitro and in vivo experiments. TrkB expression was detected in 63 (91.3%) out of 69 surgically resected primary GBC specimens by immunohistochemistry.

Ric-8A, an activator protein of Gαi, controls mammalian epithelial cell polarity for tight junction assembly and cystogenesis.

Correct cyst morphogenesis of epithelial cells requires apical-basal polarization, which is partly regulated by mitotic spindle orientation, a process dependent on the heterotrimeric G protein subunit Gαi and its binding protein LGN. Here, we show that in three-dimensional culture of mammalian epithelial Madin-Darby canine kidney (MDCK) cells, the Gαi-activating protein Ric-8A is crucial for orientation of the mitotic spindle and formation of normal cysts that comprise a single layer of polarized cells with their apical surfaces lining an inner lumen. Consistent with the involvement of LGN, cystogenesis can be well organized by ADP-ribosylated Gαi, retaining the ability to interact with LGN, but not by the interaction-defective mutant protein Gαi2 (N150I).

Ric-8A-mediated stabilization of the trimeric G protein subunit Gαi is inhibited by pertussis toxin-catalyzed ADP-ribosylation.

The heterotrimeric G protein subunit Gαi can be activated by G protein-coupled receptors and the cytosolic protein Ric-8A, the latter of which is also known to prevent ubiquitin-dependent degradation of Gαi. Here we show that the amounts of the three Gαi-related proteins Gαi1, Gαi2, and Gαi3, but not that of Gαq, are rapidly decreased by cell treatment with pertussis toxin (PTX). The decrease appears to be due to ADP-ribosylation of Gαi, because PTX treatment does not affect the amount of a mutant Gαi2 carrying alanine substitution for Cys352, the residue that is ADP-ribosylated by the toxin.

c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions.

Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1.

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys⁴⁰ in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys⁴⁰ acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood.

The cell polarity protein mInsc regulates neutrophil chemotaxis via a noncanonical G protein signaling pathway.

Successful chemotaxis requires not only increased motility but also sustained directionality. Here, we show that, during neutrophil chemotaxis via receptors coupled with the Gi family of heterotrimeric G proteins, directional movement is regulated by mInsc, a mammalian protein distantly related to the Drosophila polarity-organizer Inscuteable. The GDP-bound, Gβγ-free Gαi subunit accumulates at the front of chemotaxing neutrophils to recruit mInsc-complexed with the Par3-aPKC evolutionarily conserved polarity complex-via LGN/AGS3 that simultaneously binds to Gαi-GDP and mInsc.

Ubiquitination of the heterotrimeric G protein α subunits Gαi2 and Gαq is prevented by the guanine nucleotide exchange factor Ric-8A.

The cytosolic protein Ric-8A acts as a guanine nucleotide exchange factor for Gα subunits of the Gi, Gq, and G12/13 classes of heterotrimeric G protein in vitro, and is also known to increase the amounts of these Gα proteins in vivo. The mechanism whereby Ric-8 regulates Gα content, however, has not been fully understood. Here we show that Ric-8 Astabilizes Gαi2 and Gαq by preventing their ubiquitination.

The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells.

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.

Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment.

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro.

Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression.

Most cells in tissues are polarized and usually have two distinct plasma membrane domains-an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin-Darby canine kidney II cells as a model of polarized cells.

Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN).

Interaction between the mammalian cell polarity proteins mInsc (mammalian homologue of Inscuteable) and Leu-Gly-Asn repeat-enriched protein (LGN), as well as that between their respective Drosophila homologues Inscuteable and Partner of Inscuteable (Pins), plays crucial roles in mitotic spindle orientation, a process contributing to asymmetric cell division. Here, we report a crystal structure of the LGN-binding domain (LBD) of human mInsc complexed with the N-terminal tetratricopeptide repeat (TPR) motifs of human LGN at 2.6-Å resolution.

Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex.

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown.

Membrane phospholipid metabolism during phagocytosis in human neutrophils.

Neutrophils play an essential role via phagocytosis in host defense against microbial infections. However, little is known about molecular mechanisms underlying phagocytosis in neutrophils, because of the difficulty in genetically manipulating these cells. Here, we provide the first comprehensive description of phospholipid metabolism during phagocytosis in human neutrophils, which we have efficiently transfected with cDNAs encoding lipid-probing protein modules.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase.

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production.

Structure and function of the PB1 domain, a protein interaction module conserved in animals, fungi, amoebas, and plants.

Proteins containing the PB1 domain, a protein interaction module conserved in animals, fungi, amoebas, and plants, participate in diverse biological processes. The PB1 domains adopt a ubiquitin-like beta-grasp fold, containing two alpha helices and a mixed five-stranded beta sheet, and are classified into groups harboring an acidic OPCA motif (type I), the invariant lysine residue on the first beta strand (type II), or both (type I/II). The OPCA motif of a type I PB1 domain forms salt bridges with basic residues, especially the conserved lysine, of a type II PB1 domain, thereby mediating a specific PB1-PB1 heterodimerization, whereas additional contacts contribute to high affinity and specificity of the modular interaction.

Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding.

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro.

Two forms of human Inscuteable-related protein that links Par3 to the Pins homologues LGN and AGS3.

In cell polarization of Drosophila neuroblasts, Inscuteable (Insc) functions via tethering Partner of Insc (Pins) to Bazooka, homologous to human cell polarity protein Par3. However, little has been known about mammalian homologues of Insc. Here we describe cloning of two distinct cDNAs from human Insc gene, which is differentially expressed from alternative first exons: one encodes 579 amino acids, whereas the other lacks the N-terminal 47 amino acids.