scholarly journals Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox

1996 ◽  
Vol 317 (3) ◽  
pp. 919-924 ◽  
Author(s):  
Frans B. WIENTJES ◽  
George PANAYOTOU ◽  
Emer REEVES ◽  
Anthony W. SEGAL
Keyword(s):  
Nadph Oxidase ◽  
Binding Assays ◽  
Phagocytic Cells ◽  
Sh3 Domains ◽  
Cytosolic Proteins ◽  
Src Homology 3 ◽  
Vitro Binding ◽  
Src Homology ◽  
Flavocytochrome B

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.

scholarly journals p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains

1993 ◽  
Vol 296 (3) ◽  
pp. 557-561 ◽  
Author(s):  
F B Wientjes ◽  
J J Hsuan ◽  
N F Totty ◽  
A W Segal
Keyword(s):  
Nadph Oxidase ◽  
Sequence Similarity ◽  
Granulomatous Disease ◽  
Phagocytic Cells ◽  
Src Homology 3 ◽  
N Terminus ◽  
Phagocyte Oxidase ◽  
Src Homology ◽  
Membrane Bound ◽  
Flavocytochrome B

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.

scholarly journals CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells.

1996 ◽  
Vol 183 (1) ◽  
pp. 179-186 ◽  
Author(s):  
R Galandrini ◽  
G Palmieri ◽  
M Piccoli ◽  
L Frati ◽  
A Santoni
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Tyrosine Phosphorylation ◽  
Adaptor Proteins ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Vitro Binding ◽  
Human Natural Killer Cells ◽  
Src Homology

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.

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

2002 ◽  
Vol 83 (12) ◽  
pp. 3147-3152 ◽  
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.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP

2001 ◽  
Vol 280 (2) ◽  
pp. C248-C253 ◽  
Author(s):  
Stephen C. Dahl ◽  
Joseph S. Handler ◽  
H. Moo Kwon
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
Kinase Inhibitors ◽  
Dependent Manner ◽  
Binding Assays ◽  
P38 Inhibitor ◽  
Vitro Binding ◽  
Compatible Osmolytes

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

KPNA7, an oocyte- and embryo-specific karyopherin?subtype, is required for porcine embryo development

2012 ◽  
Vol 24 (2) ◽  
pp. 382 ◽  
Author(s):  
Xin Wang ◽  
Ki-Eun Park ◽  
Stephanie Koser ◽  
Shihong Liu ◽  
Luca Magnani ◽  
...  
Keyword(s):  
Binding Assays ◽  
Control Groups ◽  
Nuclear Localisation ◽  
Cytoplasmic Proteins ◽  
Vitro Binding ◽  
Intracellular Proteins ◽  
Porcine Embryo ◽  
Stage Embryo ◽  
Interfering Rna

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P < 0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherin α subtypes and that these karyopherin α subtypes differentially transport intracellular cargo during cleavage development.

The Ku70 autoantigen interacts with p40phox in B lymphocytes

1999 ◽  
Vol 112 (4) ◽  
pp. 503-513
Author(s):  
N. Grandvaux ◽  
S. Grizot ◽  
P.V. Vignais ◽  
M.C. Dagher
Keyword(s):  
Nadph Oxidase ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Cell Extract ◽  
Dependent Protein ◽  
Terminal Amino ◽  
Flavocytochrome B ◽  
Lymphocyte Cell ◽  
Two Hybrid

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50–260) and the C-terminal extremity (aa 260–339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

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