Sachiko Kamakura

Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling.

Although the Notch and JAK–STAT signalling pathways fulfill overlapping roles in growth and differentiation regulation, no coordination mechanism has been proposed to explain their relationship. Here we show that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5. Hes proteins associate with JAK2 and STAT3, and facilitate complex formation between JAK2 and STAT3, thus promoting STAT3 phosphorylation and activation. Furthermore, suppression of endogenous Hes1 expression reduces growth factor induction of STAT3 phosphorylation. STAT3 seems to be essential for maintenance of radial glial cells and differentiation of astrocytes by Notch in the developing central nervous system. These results suggest that direct protein–protein interactions coordinate cross-talk between the Notch–Hes and JAK–STAT pathways.

Distinct Domains of Mouse Dishevelled Are Responsible for the c-Jun N-terminal Kinase/Stress-activated Protein Kinase Activation and the Axis Formation in Vertebrates

Recent studies have shown thatDrosophila Dishevelled (Dsh), an essential component of thewingless signal transduction, is also involved in planar polarity signaling through the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway inDrosophila. Here, we show that expression of a mouse homolog of Dsh (mDvl-1) in NIH3T3 cells activates JNK/SAPK, and its activator MKK7. A C-terminal half of mDvl-1 which contains the DEP domain was sufficient for the activation of JNK/SAPK, whereas an N-terminal half of mDvl-1 as well as the DEP domain is required for stimulation of the TCF/LEF-1-dependent transcriptional activation, a β-catenin-dependent process. A single amino acid substitution (Met for Lys) within the DEP domain (mDvl-1 (KM)) abolished the JNK/SAPK-activating activity of mDvl-1, but did not affect the activity to activate the LEF-1-dependent transcription. Ectopic expression of mDvl-1 (KM) or an N-terminal half of mDvl-1, but not the C-terminal, was able to induce secondary axis inXenopus embryos. Because the secondary axis formation is dependent on the Wnt/β-catenin signaling pathway, these results suggest that distinct domains of mDvl-1 are responsible for the two downstream signaling pathways, the β-catenin pathway and the JNK/SAPK pathway in vertebrates.

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 β-grasp fold, containing two α helices and a mixed five-stranded β sheet, and are classified into groups harboring an acidic OPCA motif (type I), the invariant lysine residue on the first β 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. The canonical PB1 dimerization is required for the formation of complexes between p40phox and p67phox (for activation of the NADPH oxidase crucial for mammalian host defense), between the scaffold Bem1 and the guanine nucleotide exchange factor Cdc24 (for polarity establishment in yeasts), and between the polarity protein Par6 and atypical protein kinase C (for cell polarization in animal cells), as well as for the interaction between the mitogen-activated protein kinase kinase kinases MEKK2 or MEKK3 and the downstream target mitogen-activated protein kinase kinase MEK5 (for early cardiovascular development in mammals). PB1 domains can also mediate interactions with other protein domains. For example, an intramolecular interaction between the PB1 and PX domains of p40phox regulates phagosomal targeting of the microbicidal NADPH oxidase; the PB1 domain of MEK5 is likely responsible for binding to the downstream kinase ERK5, which lacks a PB1 domain; and the scaffold protein Nbr1 associates through a PB1-containing region with titin, a sarcomere protein without a PB1 domain. This Review describes various aspects of PB1 domains at the molecular and cellular levels. With 9 figures, four interactive structure figures, and 170 citations, this Review describes the structure-function relationships of proteins with PB1 domains. Three types of PB1-containing proteins occur: those with a type I domain, those with a type II domain, and those with both type I and type II (type I/II). The type I domain mediates interactions with proteins containing a type II domain in a canonical PB1-PB1 interaction. Interactions mediated by PB1 domains are important for organizing cell structure, for example, in polarized cells (epithelial cells and neurons) and in skeletal muscle. In addition, interactions involving PB1 domains influence the activation of mitogen-activated protein kinase signaling and activation of NADPH oxidase.

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. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae ΔbemA mutant was defective in hyphal morphogenesis and growth in culture and in planta. The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δcdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.

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. The crystal structure of the full‐length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1‐mediated PX regulation occurs without preventing the PB1–PB1 association with p67phox.

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. Both mInsc-deficient and aPKC-blocked neutrophils exhibit a normal motile activity but migrate in an undirected manner. mInsc deficiency prevents neutrophils from efficiently stabilizing pseudopods at the leading edge; the stability is restored by wild-type mInsc, but not by a mutant protein defective in binding to LGN/AGS3. Thus, mInsc controls directional migration via noncanonical G protein signaling, in which Gβγ-free Gαi-GDP, a product from Gαi-GTP released after receptor activation, plays a central role.

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. In the complex, mInsc-LBD adopts an elongated structure with three binding modules—an α-helix, an extended region, and a β-sheet connected with a loop—that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPRs. Structural analysis and structure-based mutagenesis define residues that are critical for mInsc–LGN association, and reveal that the activator of G-protein signaling 3 (AGS3)-binding protein Frmpd1 [4.1/ezrin/radixin/moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein 1] and its relative Frmpd4 interact with LGN via a region homologous to a part of mInsc-LBD, whereas nuclear mitotic apparatus protein (NuMA) and the C terminus of LGN recognize the TPR domain in a manner different from that by mInsc. mInsc binds to LGN with the highest affinity (KD ≈ 2.4 nM) and effectively replaces the Frmpd proteins, NuMA, and the LGN C terminus, suggesting the priority of mInsc in binding to LGN. We also demonstrate, using mutant proteins, that mInsc–LGN interaction is vital for stabilization of LGN and for intracellular localization of mInsc.

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. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

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

Par6–aPKC recruitment to the premature apical membrane through Morg1 interaction with Par6 is required for definition of apical identity of epithelial cells.

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

Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in MDCK II cells in a Rab27A-dependent manner. Apical membrane localization of podocalyxin is required for expression of the tight junction protein claudin-2 through modulation of intracellular signals, including MAPK signals.