The Experts below are selected from a list of 1743 Experts worldwide ranked by ideXlab platform
Robert V. Stahelin - One of the best experts on this subject based on the ideXlab platform.
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Stahelin et al 1 Structural and Membrane Binding Analysis of the PX Domain of
2015Co-Authors: Phosphoinositide -kinase-cα, Robert V. Stahelin, Karol S. Bruzik, Dimitrios Karathanassis, Michael D. Waterfield, Roger L. Williams, Wonhwa ChoAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX Domains, we determined the crystal structure of the PX Domain from phosphoinositide 3-kinase C2α (PI3K-C2α), which binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). To delineate the mechanism by which this PX Domain interacts with membranes, we measured the membrane binding of the wild type Domain and mutants by surface plasmon resonance and monolaye
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p47phox phox homology Domain regulates plasma membrane but not phagosome neutrophil nadph oxidase activation
Journal of Biological Chemistry, 2010Co-Authors: Xing Jun Li, Christophe C Marchal, Natalie D Stull, Robert V. Stahelin, Mary C. DinauerAbstract:Abstract The assembly of cytosolic subunits p47phox, p67phox, and p40phox with flavocytochrome b558 at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47phox Phox homology (PX) Domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47phox PX Domain in neutrophils is unclear, we investigated its role using p47phox knock-out (KO) mouse neutrophils to express human p47phox and derivatives harboring R90A mutations in the PX Domain that result in loss of phosphoinositide binding. Human p47phox proteins were expressed at levels similar to endogenous murine p47phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47phox rescued p47phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47phox with Arg90 substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47phox Arg90 mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47phox PX Domain for neutrophil NADPH oxidase activation.
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Structural and membrane binding analysis of the Phox homology Domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity.
The Journal of biological chemistry, 2007Co-Authors: Robert V. Stahelin, Diana Murray, Dimitrios Karathanassis, Roger Williams, Wonhwa ChoAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX Domains, we determined the crystal structure of the PX Domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX Domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX Domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX Domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX Domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the Domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX Domains, the Bem1p PX Domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX Domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX Domains.
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Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α
The Journal of biological chemistry, 2006Co-Authors: Robert V. Stahelin, Karol S. Bruzik, Jerónimo Bravo, Dimitrios Karathanassis, Michael D. Waterfield, Roger L. WilliamsAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX Domains, we determined the crystal structure of the PX Domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX Domain interacts with membranes, we measured the membrane binding of the wild type Domain and mutants by surface plasmon resonance and monolayer techniques. This PX Domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX Domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX Domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 Domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX Domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX Domains and elucidate the mechanism by which the PI3K-C2alpha PX Domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.
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Molecular Mechanism of Membrane Docking by the Vam7p PX Domain
The Journal of biological chemistry, 2006Co-Authors: Stephanie A. Lee, Robert V. Stahelin, James M. Kovacs, Matthew L Cheever, Thanuja Gangi Setty, Christopher G. BurdAbstract:The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX Domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX Domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX Domain association with PtdIns(3)P-containing membranes. The inserting Val70, Leu71, and Trp75 residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX Domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX Domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.
Dimitrios Karathanassis - One of the best experts on this subject based on the ideXlab platform.
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Stahelin et al 1 Structural and Membrane Binding Analysis of the PX Domain of
2015Co-Authors: Phosphoinositide -kinase-cα, Robert V. Stahelin, Karol S. Bruzik, Dimitrios Karathanassis, Michael D. Waterfield, Roger L. Williams, Wonhwa ChoAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX Domains, we determined the crystal structure of the PX Domain from phosphoinositide 3-kinase C2α (PI3K-C2α), which binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). To delineate the mechanism by which this PX Domain interacts with membranes, we measured the membrane binding of the wild type Domain and mutants by surface plasmon resonance and monolaye
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Structural and membrane binding analysis of the Phox homology Domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity.
The Journal of biological chemistry, 2007Co-Authors: Robert V. Stahelin, Diana Murray, Dimitrios Karathanassis, Roger Williams, Wonhwa ChoAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX Domains, we determined the crystal structure of the PX Domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX Domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX Domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX Domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX Domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the Domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX Domains, the Bem1p PX Domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX Domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX Domains.
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Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α
The Journal of biological chemistry, 2006Co-Authors: Robert V. Stahelin, Karol S. Bruzik, Jerónimo Bravo, Dimitrios Karathanassis, Michael D. Waterfield, Roger L. WilliamsAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX Domains, we determined the crystal structure of the PX Domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX Domain interacts with membranes, we measured the membrane binding of the wild type Domain and mutants by surface plasmon resonance and monolayer techniques. This PX Domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX Domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX Domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 Domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX Domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX Domains and elucidate the mechanism by which the PI3K-C2alpha PX Domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.
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structural and membrane binding analysis of the PX Domain of phosphoinositide 3 kinase c2α
2006Co-Authors: Robert V. Stahelin, Karol S. Bruzik, Dimitrios Karathanassis, Michael D. Waterfield, Roger L. Williams, Melchor Fernández AlmagroAbstract:Phox homology (PX) Domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX Domains, we determined the crystal structure of the PX Domain from phosphoinositide 3-kinase C2α (PI3K-C2α), which binds phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2). To delineate the mechanism by which this PX Domain interacts with membranes, we measured the membrane binding of the wild type Domain and mutants by surface plasmon resonance and monolayer techniques. This PX Domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P2 binding. The membrane binding of the PX Domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P2. Furthermore, the PX Domain displayed significantly higher PtdIns(4,5)P2 membrane affinity and specificity when compared to the PI3K-C2α C2 Domain, demonstrating that high affinity PtdIns(4,5)P2 binding was facilitated by the PX Domain in fulllength PI3K-C2α . Together, these studies provide new structural insight into the diverse PI specificities of PX Domains and elucidate the mechanism by which the PI3K-C2α PX Domain interacts with PtdIns(4,5)P2-containing membranes and thereby mediates the membrane recruitment of PI3K-C2α .
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binding of the PX Domain of p47phox to phosphatidylinositol 3 4 bisphosphate and phosphatidic acid is masked by an intramolecular interaction
The EMBO Journal, 2002Co-Authors: Dimitrios Karathanassis, Robert V. Stahelin, Jerónimo Bravo, Christine M Pacold, Olga Perisic, Roger WilliamsAbstract:p47phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47phox PX Domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40phox, while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47phox show that membrane interaction by the PX Domain is masked by an intramolecular association with the C-terminal SH3 Domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX Domain.
Wanjin Hong - One of the best experts on this subject based on the ideXlab platform.
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deficiency of sorting nexin 27 snx27 leads to growth retardation and elevated levels of n methyl d aspartate receptor 2c nr2c
Molecular and Cellular Biology, 2011Co-Authors: Lei Cai, Li Shen Loo, Vadim Atlashkin, Brendon J Hanson, Wanjin HongAbstract:Phox (PX) Domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) Domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX Domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ Domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27−/− neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.
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The Phox (PX) Domain proteins and membrane traffic.
Biochimica et biophysica acta, 2006Co-Authors: Li-fong Seet, Wanjin HongAbstract:Phosphoinositides (PIs) are phosphorylated derivatives of phosphatidylinositol (PtdIns) that regulate many cellular and physiological processes. Most PIs act by serving as membrane docking sites for proteins harboring specific PI-binding Domains so that the location and function of these proteins could be dynamically governed. The Phox (PX) Domain represents a novel PI-binding module capable of regulating membrane targeting of about 47 mammalian proteins, 30 of which are tentatively referred to as sorting nexins (SNXs). Some SNXs have been implicated in regulating membrane trafficking in the endocytic pathway. We discuss here recent development and progress in the study of the PX Domain-containing proteins.
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human nischarin imidazoline receptor antisera selected protein is targeted to the endosomes by a combined action of a PX Domain and a coiled coil region
Journal of Biological Chemistry, 2004Co-Authors: Koh-pang Lim, Wanjin HongAbstract:Abstract Around 50 mammalian and 15 yeast proteins are known to contain the phox (PX) Domain, the majority (about 30) of which is classified as sorting nexins (SNXs). The PX Domain, a hallmark of these proteins, is a conserved stretch of about 120 amino acids and is recently shown to mediate phosphoinositide binding. A few PX Domain proteins (including some SNXs) have been shown to participate in diverse cellular processes such as protein sorting, signal transduction, and vesicle fusion. In this report, we present our results supporting a role of human IRAS to act as a SNX. The mouse homologue, previously identified as Nischarin, has been shown to interact with the α5 subunit of integrin and inhibit cell migration (Alahari, S. K., Lee J. W., and Juliano R. L. (2000) J. Cell Biol. 51, 1141-1154). Its human homologue (imidazoline receptor antisera-selected (IRAS)), on the other hand, contains an NH2-terminal extension and is a larger protein of 1504 amino acids consisting of an NH2-terminal PX Domain, 5 putative leucine-rich repeats, a predicted coiled-coil Domain, and a long COOH-terminal region. We show that it has the ability to homo-oligomerize via its coiled-coil region. The PX Domain of IRAS is essential for association with phosphatidylinositol 3-phosphate-enriched endosomal membranes. However, the PX Domain of IRAS alone is insufficient for its localization to endosomes, unless the coiled-coil Domain was included or it is artificially dimerized by glutathione S-transferase. Interaction of human IRAS with α5 integrin is not affected by the NH2-terminal extension, and overexpression of IRAS could cause a redistribution of surface α5 integrin to intracellular endosomal structures.
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The Phox homology (PX) Domain, a new player in phosphoinositide signalling.
Biochemical Journal, 2001Co-Authors: Li-fong Seet, Brendon Hanson, Wanjin HongAbstract:Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) Domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE Domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) Domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX Domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) Domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.
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snx3 regulates endosomal function through its PX Domain mediated interaction with ptdins 3 p
Nature Cell Biology, 2001Co-Authors: Heinz Hortsman, Li-fong Seet, Siew Heng Wong, Wanjin HongAbstract:The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX Domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.
Chris D. Ellson - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40phox in Activation of the Neutrophil NADPH Oxidase
The Journal of biological chemistry, 2007Co-Authors: Sarah A. Bissonnette, Chris D. Ellson, Christina M. Glazier, Mary Q. Stewart, Glenn E. Brown, Michael B. YaffeAbstract:In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX Domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX Domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) Domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 Domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 Domain engagement.
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PtdIns3P binding to the PX Domain of p40phox is a physiological signal in NADPH oxidase activation
The EMBO journal, 2006Co-Authors: Chris D. Ellson, Karen E. Anderson, Len R Stephens, Keith Davidson, Phillip T. HawkinsAbstract:The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX Domains of two oxidase components, p47phox and p40phox, are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX Domain of their p40phox gene, which selectively prevents binding to PtdIns3P. p40phoxR58A/R58A embryos do not develop normally but p40phoxR58A/− mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40phox+/− siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40phox. p40phoxR58A/− mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.
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The PX Domain: a new phosphoinositide-binding module.
Journal of cell science, 2002Co-Authors: Chris D. Ellson, Simon Andrews, Len R Stephens, Phill T HawkinsAbstract:The PX Domain, which until recently was an orphan Domain, has emerged as the latest member of the phosphoinositide-binding module superfamily. Structural studies have revealed that it has a novel fold and identified key residues that interact with the bound phosphoinositide, enabling some prediction of phosphoinositide-binding specificity. Specificity for PtdIns(3)P appears to be the most common, and several proteins containing PX Domains localise to PtdIns(3)P-rich endosomal and vacuolar structures through their PX Domains: these include the yeast t-SNARE Vam7p, mammalian sorting nexins (involved in membrane trafficking events) and the Ser/Thr kinase CISK, which is implicated in cell survival. Additionally, phosphoinositide binding to the PX Domains of p40(phox) and p47(phox) appears to play a critical role in the active assembly of the neutrophil oxidase complex.
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PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX Domain of p40(phox).
Nature Cell Biology, 2001Co-Authors: Chris D. Ellson, S. Gobert-gosse, K. E. Anderson, K. Davidson, H. Erdjument-bromage, P. Tempst, J. W. Thuring, M. A. Cooper, Z. Y. Lim, A. B. HolmesAbstract:The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX Domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX Domain.
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The Crystal Structure of the PX Domain from P40Phox Bound to Phosphatidylinositol 3-Phosphate
Molecular cell, 2001Co-Authors: Jerónimo Bravo, Dimitrios Karathanassis, Christine M Pacold, Michael E. Pacold, Chris D. Ellson, Karen E. Anderson, P. Jonathan G. Butler, Isabelle Lavenir, Olga Perisic, Phillip T. HawkinsAbstract:More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX Domain. The 1.7 A X-ray crystal structure of the PX Domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX Domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX Domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 Domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX Domain.
Hideki Sumimoto - One of the best experts on this subject based on the ideXlab platform.
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atypical membrane embedded phosphatidylinositol 3 4 bisphosphate pi 3 4 p2 binding site on p47phox phox homology PX Domain revealed by nmr
Journal of Biological Chemistry, 2012Co-Authors: Pavlos Stampoulis, Hideki Sumimoto, Takumi Ueda, Masahiko Matsumoto, Hiroaki Terasawa, Kei Miyano, Ichio ShimadaAbstract:The Phox homology (PX) Domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47phox PX Domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P2 binding mode of the p47phox PX Domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P2-binding site, which includes the residues of helices α1 and α1′ and the following loop up to the distorted left-handed PPII helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX Domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47phox PX Domain suggest that different types of phosphoinositides sequentially bind to the p47phox PX Domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.
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phosphorylation of p47phox directs phox homology Domain from sh3 Domain toward phosphoinositides leading to phagocyte nadph oxidase activation
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Tetsuro Ago, Daisuke Kohda, Hidekazu Hiroaki, Ryu Takeya, Futoshi Kuribayashi, Takashi Ito, Hideki SumimotoAbstract:Protein–phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47phox, a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47phox with phosphoinositides. Although the isolated phox homology (PX) Domain of p47phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX Domain with the C-terminal Src homology 3 (SH3) Domain, and hence the wild-type full-length p47phox is incapable of binding to the lipids. The W263R substitution in this SH3 Domain, abrogating the interaction with the PX Domain, leads to a binding of p47phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX Domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47phox, because protein kinase C treatment of the wild-type p47phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47phox nor lipid-binding-defective proteins, one lacking the PX Domain and the other carrying the R90K substitution in this Domain, migrates. Thus the protein phosphorylation-driven conformational change of p47phox enables its PX Domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.
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The PX Domain as a Novel Phosphoinositide- Binding Module
Biochemical and biophysical research communications, 2001Co-Authors: Tetsuro Ago, Daisuke Kohda, Hidekazu Hiroaki, Ryu Takeya, Futoshi Kuribayashi, Takashi Ito, Hideki SumimotoAbstract:Abstract The phox (phagocyte oxidase) homology (PX) Domain occurs in the mammalian phox proteins p40phox and p47phox, the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX Domains of p40phox and p47phox directly bind to phosphoinositides: p40phox prefers Ptdlns(3)P, while p47phox does Ptdlns(4)P and Ptdlns(3,4)P2. In addition, the Bem1p PX Domain also interacts with Ptdlns(4)P. When the p40phox PX Domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40phox PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX Domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.
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solution structure of the PX Domain a target of the sh3 Domain
Nature Structural & Molecular Biology, 2001Co-Authors: Hidekazu Hiroaki, Hideki Sumimoto, Daisuke KohdaAbstract:The phox homology (PX) Domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX Domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 Domain derived from the same protein. The solution structure of p47 PX has an α + β structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 Domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.
