A new role of Pro-73 of p47phox in the activation of neutrophil NADPH oxidase

doi:10.1016/S0003-9861(03)00296-0

Archives of Biochemistry and Biophysics

Volume 416, Issue 1, 1 August 2003, Pages 92-100

https://doi.org/10.1016/S0003-9861(03)00296-0 Get rights and content

Abstract

The PX domain of p47^phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47^phox(1–286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67^phox(1–210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b₅₅₈.

Section snippets

Materials

Glutathione–Sepharose, CM-Sepharose FF, pGEX-6P, and Escherichia coli BL21 strain were purchased from Amersham Biosciences (Piscataway, NJ). Oligonucleotide primers for PCR and mutations were synthesized by the same manufacturer. BamHI and EcoRI were obtained from Toyobo (Tokyo, Japan). Thrombin and cytochrome c were purchased from Sigma–Aldrich (St. Louis, MO). Diisopropyl fluorophosphate was obtained from Wako Pure Chemicals (Osaka, Japan). GTP and FAD were purchased from Nacalai Tesque,

Requirement of PX domain for activation

Two groups reported that deletion of PX domain diminishes the ability of activation [18], [19]. However, as their deletions included additional 25 amino acid residues following the PX domain, actual role of the PX domain has not been clarified. Here, we made a p47^phox fragment that lacks the exact region of the PX domain (assigned as ΔPX) and examined its ability to activate (Table 1). ΔPX showed a considerably lower ability to activate (33%). When the deletion was extended to residue 153

Discussion

A truncated form of p47^phox (1–286) (p47N) is able to produce full activation of NADPH oxidase in a cell-free reconstitution system [19], [20], indicating that p47N contains all the domains essential for the activation. The p47N fragment possesses a PX domain and two SH3 domains with a connecting region in-between. The SH3 domains are thought to be involved in interaction with p22^phox when unmasked. The PX domain is reportedly involved in negative regulation by interacting with one of the

Acknowledgements

This work was supported in part by a grant (13480297) for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. We are grateful to Shuhei Kobayashi, Mamoru Tanaka, Yasuaki Komeima, Tetsutaro Kai, and Kyoji Watanabe for their excellent technical assistance and to Drs. Koichi Tanaka, Yaeta Endo, and Keiichi Kato (Department of Applied Chemistry, Ehime University) for their discussions and instruments. We are grateful to Drs. Chang-Hoon Han (Department of

References (48)

B.M Babior
Blood
(1984)
F Rossi
Biochim. Biophys. Acta
(1986)
P Bellavite
Free Radic. Biol. Med.
(1988)
A.J Thrasher et al.
Biochim. Biophys. Acta
(1994)
B.M Babior
Blood
(1999)
M Tamura et al.
Biochim. Biophys. Acta
(2001)
M Tamura et al.
Biochem. Biophys. Res. Commun.
(2000)
I de Mendez et al.
J. Biol. Chem.
(1994)
P Finan et al.
J. Biol. Chem.
(1994)
F.R DeLeo et al.
J. Biol. Chem.
(1996)

T Ito et al.

FEBS Lett.

(1996)

K Hata et al.

J. Biol. Chem.

(1998)

K Ebisu et al.

J. Biol. Chem.

(2001)

C.H Han et al.

J. Biol. Chem.

(1998)

A Shiose et al.

J. Biol. Chem.

(2000)

T Ago et al.

Biochem. Biophys. Res. Commun.

(2001)

Y Zhan et al.

J. Biol. Chem.

(2002)

M Tamura et al.

J. Biol. Chem.

(1992)

H Fujii et al.

Biochim. Biophys. Acta

(1992)

M Tamura et al.

J. Biol. Chem.

(1988)

D Rotrosen et al.

J. Biol. Chem.

(1990)

A Nakanishi et al.

J. Biol. Chem.

(1992)

A.R Cross et al.

J. Biol. Chem.

(1995)

L.S Yoshida et al.

J. Biol. Chem.

(1998)

Cited by (7)

Atypical membrane-embedded phosphatidylinositol 3,4-bisphosphate (PI(3,4)P<inf>2</inf>)-binding site on p47phox phox homology (PX) domain revealed by NMR
2012, Journal of Biological Chemistry
Citation Excerpt :
Interestingly, our results can provide a rationale for the in vivo behaviors of two mutants, those of Pro-73 and Arg-70, that are within the presently identified C16-PI(3,4)P2-containing membrane-interacting interface. It has been reported that the mutation Pro-73 abrogates phosphoinositide recognition and interferes with the in vitro activity of NADPH oxidase (46), while the mutation Arg-70 prevents membrane translocation in HEK293 cells (47). Our experiments revealed that PA and soluble C4-PI(3,4)P2 competitively bind to the p47phox domain (Fig. 4).
The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47^phox 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 p47^phox 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 PP_II 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 p47^phox PX domain suggest that different types of phosphoinositides sequentially bind to the p47^phox PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.
Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages
2009, Fish and Shellfish Immunology
In vertebrates, the generation of superoxide reactive oxygen species (ROS) via activation of the Nox/Duox family of NADPH oxidases is a prototypical feature of the pathogen-induced defensive responses of activated professional phagocytes. To understand the role of the rainbow trout (Oncorhynchus mykiss) Phox oxidase from a phylogenetic and functional perspective we describe the cloning, sequencing and expression analysis of multiple NADPH components in cultured macrophages. Phylogenetic analyses support the notion of the emergence of Phox-related components before the diversification of basal euteleosts and add to the limited collection of teleost NADPH oxidases. Expression studies using lipopolysaccharide, polyinosine-polycytidylic acid and zymosan to mimic the onset of inflammatory responses in trout macrophages suggest differences in regulation of the NADPH complex throughout the maturation/differentiation period of culture and between different treatments.
The role of phosphoinositides and phosphorylation in regulation of NADPH oxidase
2004, Advances in Enzyme Regulation
Mutations in the PX-SH3<inf>A</inf> linker of p47phox decouple PI(3,4)P<inf>2</inf> binding from NADPH oxidase activation
2008, Biochemistry
Characterization of surface structure and p41phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b
2007, Biochemistry
Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes
2007, BMC Evolutionary Biology

View all citing articles on Scopus

View full text

Article preview

Archives of Biochemistry and Biophysics

Abstract

Section snippets

Materials

Requirement of PX domain for activation

Discussion

Acknowledgements

References (48)

Blood

Biochim. Biophys. Acta

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Blood

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cited by (7)

Atypical membrane-embedded phosphatidylinositol 3,4-bisphosphate (PI(3,4)P<inf>2</inf>)-binding site on p47<sup>phox</sup> phox homology (PX) domain revealed by NMR

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages

The role of phosphoinositides and phosphorylation in regulation of NADPH oxidase

Mutations in the PX-SH3<inf>A</inf> linker of p47<sup>phox</sup> decouple PI(3,4)P<inf>2</inf> binding from NADPH oxidase activation

Characterization of surface structure and p41<sup>phox</sup> SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b

Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes