Role of Src homology 3 (SH3) domains in activation of the neutrophil NADPH oxidase

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.12.5345 ◽

1994 ◽

Vol 91 (12) ◽

pp. 5345-5349 ◽

Cited By ~ 172

Author(s):

H. Sumimoto ◽

Y. Kage ◽

H. Nunoi ◽

H. Sasaki ◽

T. Nose ◽

...

Keyword(s):

Nadph Oxidase ◽

Src Homology 3 ◽

Phagocyte Nadph Oxidase ◽

Src Homology

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Role of the Conserved Acidic Residue Asp21 in the Structure of Phosphatidylinositol 3-Kinase Src Homology 3 Domain: Circular Dichroism and Nuclear Magnetic Resonance Studies†

Biochemistry ◽

10.1021/bi001607v ◽

2001 ◽

Vol 40 (1) ◽

pp. 119-129 ◽

Cited By ~ 4

Author(s):

Nobuyuki Okishio ◽

Toshiyuki Tanaka ◽

Ryuji Fukuda ◽

Masako Nagai

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Phosphatidylinositol 3 Kinase ◽

Magnetic Resonance Studies ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology ◽

Nuclear Magnetic ◽

Phosphatidylinositol 3

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

Journal of Experimental Medicine ◽

10.1084/jem.184.3.893 ◽

1996 ◽

Vol 184 (3) ◽

pp. 893-902 ◽

Cited By ~ 64

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.

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Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer’s disease–like scenario

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0001 ◽

2014 ◽

Vol 92 (3) ◽

pp. 219-225 ◽

Cited By ~ 1

Author(s):

Kasturi Roy ◽

Oishee Chakrabarti ◽

Debashis Mukhopadhyay

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Growth Factor Receptor ◽

Adaptor Protein ◽

Sh3 Domain ◽

Binding Motifs ◽

Src Homology 3 ◽

Src Homology ◽

Cellular Compartmentalization

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2–amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer’s disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD–Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

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Intracellular targeting, interaction with Src homology 3 (SH3) domains and rolipram-detected conformational switches in cAMP-specific PDE4A phosphodiesterase

Biochemical Society Transactions ◽

10.1042/bst0250374 ◽

1997 ◽

Vol 25 (2) ◽

pp. 374-381 ◽

Cited By ~ 12

Author(s):

M. D. Houslay ◽

G. Scotland ◽

S. Erdogan ◽

E. Huston ◽

S. Mackenzie ◽

...

Keyword(s):

Sh3 Domains ◽

Src Homology 3 ◽

Intracellular Targeting ◽

Src Homology ◽

Conformational Switches

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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

Biochemical Journal ◽

10.1042/bj20021629 ◽

2003 ◽

Vol 373 (1) ◽

pp. 221-229 ◽

Cited By ~ 16

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.

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The Role of the Src Homology 3-Src Homology 2 Interface in the Regulation of Src Kinases

Journal of Biological Chemistry ◽

10.1074/jbc.m011185200 ◽

2001 ◽

Vol 276 (20) ◽

pp. 17199-17205 ◽

Cited By ~ 61

Author(s):

Stefan T. Arold ◽

Tobias S. Ulmer ◽

Terrence D. Mulhern ◽

Jörn M. Werner ◽

John E. Ladbury ◽

...

Keyword(s):

Src Kinases ◽

Src Homology 3 ◽

Src Homology 2 ◽

Src Homology

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Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains

Journal of General Virology ◽

10.1099/0022-1317-83-12-3147 ◽

2002 ◽

Vol 83 (12) ◽

pp. 3147-3152 ◽

Cited By ~ 9

Author(s):

Marita Hiipakka ◽

Kalle Saksela

Keyword(s):

Simian Immunodeficiency Virus ◽

Binding Motif ◽

Sh3 Domains ◽

Src Homology 3 ◽

High Affinity ◽

Loop Region ◽

Immunodeficiency Virus ◽

Src Homology ◽

Hiv 1

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.

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