Phosphorylation of p47phphoxdirects phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation

Tetsuro Ago, Futoshi Kuribayashi, Hidekazu Hiroaki, Ryu Takeya, Takashi Ito, Daisuke Kohda, Hideki Sumimoto

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Abstract

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.

Original languageEnglish
Pages (from-to)4474-4479
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number8
DOIs
Publication statusPublished - Apr 15 2003

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src Homology Domains
NADPH Oxidase
Phagocytes
Phosphatidylinositols
Oxidoreductases
Phosphorylation
Lipids
Carrier Proteins
Proteins
Membranes
Protein Transport
Mutant Proteins
Protein Kinase C
Membrane Proteins
Cytoplasm
Phosphotransferases
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "Phosphorylation of p47phphoxdirects phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation",
abstract = "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.",
author = "Tetsuro Ago and Futoshi Kuribayashi and Hidekazu Hiroaki and Ryu Takeya and Takashi Ito and Daisuke Kohda and Hideki Sumimoto",
year = "2003",
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doi = "10.1073/pnas.0735712100",
language = "English",
volume = "100",
pages = "4474--4479",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
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TY - JOUR

T1 - Phosphorylation of p47phphoxdirects phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation

AU - Ago, Tetsuro

AU - Kuribayashi, Futoshi

AU - Hiroaki, Hidekazu

AU - Takeya, Ryu

AU - Ito, Takashi

AU - Kohda, Daisuke

AU - Sumimoto, Hideki

PY - 2003/4/15

Y1 - 2003/4/15

N2 - 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.

AB - 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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U2 - 10.1073/pnas.0735712100

DO - 10.1073/pnas.0735712100

M3 - Article

VL - 100

SP - 4474

EP - 4479

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 8

ER -