scholarly journals Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

2003 ◽  
Vol 373 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Guihong PENG ◽  
Jin HUANG ◽  
Mellonie BOYD ◽  
Michael E. KLEINBERG
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Free System ◽  
Cell Free System ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Phagocyte Nadph Oxidase ◽  
Px Domain ◽  
Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

scholarly journals Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.

1996 ◽  
Vol 184 (3) ◽  
pp. 893-902 ◽  
Author(s):  
S Tsunawaki ◽  
S Kagara ◽  
K Yoshikawa ◽  
L S Yoshida ◽  
T Kuratsuji ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Amino Terminal ◽  
Src Homology 3 ◽  
Phagocyte Nadph Oxidase ◽  
A Cell ◽  
Src Homology ◽  
Tight Association

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

scholarly journals Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.

1994 ◽  
Vol 91 (12) ◽  
pp. 5345-5349 ◽  
Author(s):  
H. Sumimoto ◽  
Y. Kage ◽  
H. Nunoi ◽  
H. Sasaki ◽  
T. Nose ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Src Homology 3 ◽  
Phagocyte Nadph Oxidase ◽  
Src Homology

scholarly journals ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0174909 ◽  
Author(s):  
Manuela O. Gustafsson ◽  
Dara K. Mohammad ◽  
Erkko Ylösmäki ◽  
Hyunseok Choi ◽  
Subhash Shrestha ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Sh3 Domain ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Src Homology 3 ◽  
Src Homology

scholarly journals Dynamin-2 Phosphorylation as A Critical Regulatory Target of Bin1 and GSK3α for Endocytosis in Muscle

2021 ◽  
Author(s):  
Jessica Laiman ◽  
Julie Loh ◽  
Wei-Chun Tang ◽  
Mei-Chun Chuang ◽  
Hui-Kang Liu ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Membrane Curvature ◽  
Membrane Dynamics ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Hereditary Myopathy ◽  
Src Homology ◽  
T Tubules ◽  
Endocytic Regulation ◽  
Dynamin 2

Tight regulation of endocytosis ensures accurate control of cellular signaling and membrane dynamics, which are crucial for tissue morphogenesis and functions. Mutations of Bin1 and dynamin-2 (Dyn2), proteins that generate membrane curvature and sever endocytic invaginations, respectively, cause progressive hereditary myopathy. Here, we show that Bin1 inhibits Dyn2 via direct interaction of its SRC Homology 3 (SH3) domain with the proline-rich domain (PRD) of Dyn2. Phosphorylation of S848 of Dyn2 by GSK3α, a kinase downstream of insulin signaling, relieves Dyn2 from the inhibition of Bin1 and promotes endocytosis in muscle. Mutations of Bin1 associated with centronuclear myopathy disrupt its inhibition of Dyn2, thereby exaggerating Dyn2 fission activity and causing excessive fragmentation of T-tubules in the muscle cells. Our work reveals how Bin1-Dyn2 interaction fine-tunes membrane remodeling at the molecular level, and lay the foundation for future exploration of endocytic regulation and hereditary muscle diseases.

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