Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils.

ABSTRACT: p47(phox) is an essential component of the NADPH oxidase, and phosphorylation of p47(phox) is associated with activation of the enzyme. Here we have used p47(phox) affinity chromatography to extract a p47(phox) kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-beta(I), -beta(II) and -delta. The C-terminus of p47(phox) represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47(phox), we investigated the functional relevance of the interaction between PKC and p47(phox). Subcellular fractionation revealed an abnormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to particulate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47(phox) affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47(phox) can act as a regulator of PKC in neutrophils.

SUBMITTER: Reeves EP

PROVIDER: S-EPMC1220709 | biostudies-other | 1999 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Direct in vitro and in vivo evidence for interaction between Hsp47 protein and collagen triple helix.

Project description:Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.

| S-EPMC3307285 | biostudies-literature

Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

Project description:Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

| S-EPMC139531 | biostudies-literature

Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils.

Project description:Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

| S-EPMC6189209 | biostudies-literature

Statins and C-reactive protein: in silico evidence on direct interaction.

Project description:IntroductionStatins are known to lower CRP, and this reduction has been suggested to contribute to the established efficacy of these drugs in reducing cardiovascular events and outcomes. However, the exact mechanism underlying the CRP-lowering effect of statins remains elusive.MethodsIn order to test the possibility of direct interaction, we performed an in silico study by testing the orientation of the respective ligands (statins) and phosphorylcholine (the standard ligand of CRP) in the CRP active site using Molecular Operating Environment (MOE) software.ResultsDocking experiments showed that all statins could directly interact with CRP. Among statins, rosuvastatin had the strongest interaction with CRP (pKi = 16.14), followed by fluvastatin (pKi = 15.58), pitavastatin (pKi = 15.26), atorvastatin (pKi = 14.68), pravastatin (pKi = 13.95), simvastatin (pKi = 7.98) and lovastatin (pKi = 7.10). According to the above-mentioned results, rosuvastatin, fluvastatin, pitavastatin and atorvastatin were found to have stronger binding to CRP compared with the standard ligand phosphocholine (pKi = 14.55).ConclusionsThis finding suggests a new mechanism of interaction between statins and CRP that could be independent of the putative cholesterol-lowering activity of statins.

| S-EPMC7667423 | biostudies-literature

Serine-threonine kinase receptor-associated protein inhibits apoptosis signal-regulating kinase 1 function through direct interaction.

Project description:Serine-threonine kinase receptor-associated protein (STRAP) interacts with transforming growth factor beta (TGF-beta) receptors and inhibits TGF-beta signaling. Here, we identify STRAP as an interacting partner of ASK1 (apoptosis signal-regulating kinase 1). The association between ASK1 and STRAP is mediated through the C-terminal domain of ASK1 and the fourth and sixth WD40 repeats of STRAP. Using cysteine-to-serine amino acid substitution mutants of ASK1 (C1005S, C1351S, C1360S, and C1351S/C1360S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that Cys(1351) and Cys(1360) of ASK1 and Cys(152) and Cys(270) of STRAP are required for ASK1-STRAP binding. ASK1 phosphorylated STRAP at Thr(175) and Ser(179), suggesting a potential role for STRAP phosphorylation in ASK1 activity regulation. Expression of wild-type STRAP, but not STRAP mutants (C152S/C270S and T175A/S179A), inhibited ASK1-mediated signaling to both JNK and p38 kinases by stabilizing complex formation between ASK1 and its negative regulators, thioredoxin and 14-3-3, or decreasing complex formation between ASK1 and its substrate MKK3. In addition, STRAP suppressed H(2)O(2)-mediated apoptosis in a dose-dependent manner by inhibiting ASK1 activity through direct interaction. These results suggest that STRAP can act as a negative regulator of ASK1.

| S-EPMC2804202 | biostudies-literature

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Project description:Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

| S-EPMC6829599 | biostudies-literature

Identification of novel kinase inhibitors by targeting a kinase-related apoptotic protein-protein interaction network in HeLa cells.

Project description:ObjectivesProtein kinases orchestrate activation of signalling cascades in response to extra- and intracellular stimuli for regulation of cell proliferation. They are directly involved in a variety of diseases, particularly cancers. Systems biology approaches have become increasingly important in understanding regulatory frameworks in cancer, and thus may facilitate future anti-cancer discoveries. Moreover, it has been suggested and confirmed that high-throughput virtual screening provides a novel, effective way to reveal small molecule protein kinase inhibitors. Accordingly, we aimed to identify kinase targets and novel kinase inhibitors.Materials and methodsA series of bioinformatics methods, such as network construction, molecular docking and microarray analyses were performed.ResultsIn this study, we computationally constructed the appropriate global human protein-protein interaction network with data from online databases, and then modified it into a kinase-related apoptotic protein-protein interaction network. Subsequently, we identified several kinases as potential drug targets according to their differential expression observed by microarray analyses. Then, we predicted relevant microRNAs, which could target the above-mentioned kinases. Ultimately, we virtually screened a number of small molecule natural products from Traditional Chinese Medicine (TCM)@Taiwan database and identified a number of compounds that are able to target polo-like kinase 1, cyclin-dependent kinase 1 and cyclin-dependent kinase 2 in HeLa cervical carcinoma cells.ConclusionsTaken together, all these findings might hopefully facilitate discovery of new kinase inhibitors that could be promising candidates for anti-cancer drug development.

| S-EPMC6496802 | biostudies-literature

IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules.

Project description:AimsKCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K(+) channel responsible for I(Ks)-a major repolarizing current in the human heart-and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view.Methods and resultsUsing a yeast two-hybrid screening, we detected a direct interaction between beta-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of beta-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1-KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered I(Ks). In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the I(Ks) response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA-detected by phospho-specific antibodies-was maintained, as was the association of the two partners.ConclusionWe propose that the KCNQ1-KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with I(Ks) activation.

| S-EPMC2781743 | biostudies-literature

Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex.

Project description:Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.

| S-EPMC3243567 | biostudies-literature

Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1).

Project description:The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.

| S-EPMC2781587 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data