Stability of actin cytoskeleton and PKC-δ binding to actin regulate NKCC1 function in airway epithelial cells

2003 ◽  
Vol 284 (2) ◽  
pp. C487-C496 ◽  
Author(s):  
Carole M. Liedtke ◽  
Melinda Hubbard ◽  
Xiangyun Wang
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Actin Polymerization ◽  
Direct Interaction ◽  
Airway Epithelial Cells ◽  
Cell Periphery ◽  
Airway Epithelial ◽  
Protein Protein Interactions ◽  
Signaling Mechanism ◽  
Increased Activity

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-δ, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-δ were verified by Western blot analysis of immunoprecipitated proteins. PKC-δ was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-δ. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-δ. Binding of recombinant PKC-δ to NKCC1 was not detected in overlay assays. Rather, activated PKC-δ bound to actin, and this interaction was prevented by a peptide encoding δC2, a C2-like domain based on the amino acid sequence of PKC-δ. δC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-δ in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-δ indicated colocalization of PKC-δ and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-δ.

SH3 domain-containing proteins and the actin cytoskeleton in yeast

2005 ◽  
Vol 33 (6) ◽  
pp. 1247-1249 ◽  
Author(s):  
G. Mirey ◽  
A. Soulard ◽  
C. Orange ◽  
S. Friant ◽  
B. Winsor
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sh3 Domains ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

SH3 (Src homology-3) domains are involved in protein–protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)–SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott–Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

scholarly journals TRIM22. A Multitasking Antiviral Factor

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1864
Author(s):  
Isabel Pagani ◽  
Guido Poli ◽  
Elisa Vicenzi
Keyword(s):  
Protein Interactions ◽  
Influenza A ◽  
Direct Interaction ◽  
Type I Interferons ◽  
Viral Gene ◽  
Target Cells ◽  
Type I ◽  
Protein Protein Interactions ◽  
Viral Genomes ◽  
Dna And Rna

Viral invasion of target cells triggers an immediate intracellular host defense system aimed at preventing further propagation of the virus. Viral genomes or early products of viral replication are sensed by a number of pattern recognition receptors, leading to the synthesis and production of type I interferons (IFNs) that, in turn, activate a cascade of IFN-stimulated genes (ISGs) with antiviral functions. Among these, several members of the tripartite motif (TRIM) family are antiviral executors. This article will focus, in particular, on TRIM22 as an example of a multitarget antiviral member of the TRIM family. The antiviral activities of TRIM22 against different DNA and RNA viruses, particularly human immunodeficiency virus type 1 (HIV-1) and influenza A virus (IAV), will be discussed. TRIM22 restriction of virus replication can involve either direct interaction of TRIM22 E3 ubiquitin ligase activity with viral proteins, or indirect protein–protein interactions resulting in control of viral gene transcription, but also epigenetic effects exerted at the chromatin level.

scholarly journals Hormone signaling through protein destruction: a lesson from plants

2009 ◽  
Vol 296 (2) ◽  
pp. E223-E227 ◽  
Author(s):  
Xu Tan ◽  
Ning Zheng
Keyword(s):  
Protein Interactions ◽  
Hormone Receptors ◽  
Ubiquitin Ligases ◽  
Ubiquitin Proteasome System ◽  
Hormone Signaling ◽  
Protein Protein Interactions ◽  
Protein Subunit ◽  
Major Pathway ◽  
Signaling Mechanism ◽  
F Box Protein

Ubiquitin-dependent protein degradation has emerged as a major pathway regulating eukaryotic biology. By employing a variety of ubiquitin ligases to target specific cellular proteins, the ubiquitin-proteasome system controls physiological processes in a highly regulated fashion. Recent studies on a plant hormone auxin have unveiled a novel paradigm of signal transduction in which ubiquitin ligases function as hormone receptors. Perceived by the F-box protein subunit of the SCFTIR1 ubiquitin ligase, auxin directly promotes the recruitment of a family of transcriptional repressors for ubiquitination, thereby activating extensive transcriptional programs. Structural studies have revealed that auxin functions through a “molecular glue” mechanism to enhance protein-protein interactions with the assistance of another small molecule cofactor, inositol hexakisphosphate. Given the extensive repertoire of similar ubiquitin ligases in eukaryotic cells, this novel and widely adopted hormone-signaling mechanism in plants may also exist in other organisms.

Oxysophocarpine protects airway epithelial cells against inflammation and apoptosis by inhibiting miR-155 expression

2020 ◽  
Vol 12 (16) ◽  
pp. 1475-1487
Author(s):  
Linfu Li ◽  
Renbing Shi ◽  
Weimei Shi ◽  
Rui Zhang ◽  
Longhuo Wu
Keyword(s):  
Epithelial Cells ◽  
Protein Interactions ◽  
Inflammatory Responses ◽  
Annexin V ◽  
Airway Epithelial Cells ◽  
Protective Effects ◽  
Airway Epithelial ◽  
Quantitative Real Time Pcr ◽  
Protein Expressions ◽  
Anti Inflammation

Oxysophocarpine (OSC) has been documented for anti-inflammatory activity. However, the mechanisms of OSC in anti-inflammation are unclear. Aim: To investigate the protective effects of OSC on inflammation and apoptosis induced by lipopolysaccharide in NCI-H292 and human primary airway epithelial cells. Materials & methods: MTT and Annexin V-FITC/PI staining were used to detect cells viability. Inflammatory responses were determined by ELISA. The quantitative real-time PCR (qRT-PCR) and western blot were used to detect mRNA/miRNA and protein expressions respectively. Co-immunoprecipitation was investigated for protein interactions. Results & conclusion: miR-155 mimics significantly induced cell apoptosis, inflammatory responses and MAPK and NF-κB pathways. NDFIP1 was identified as the target of miR-155. OSC protected cells against apoptosis and inflammatory responses and compromised miR-155 activity by attenuating MAPK and NF-κB pathways.

C-Cbl-PI3K Interaction Is Important for Platelet Outside-In Signaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2014-2014
Author(s):  
Claudia Lorena Buitrago ◽  
Satya P. Kunapuli ◽  
Archana Sanjay
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Conflicts Of Interest ◽  
Physiological Role ◽  
Human Platelets ◽  
Scaffolding Protein ◽  
Protein Protein Interactions ◽  
Clot Retraction ◽  
Knock Out

Abstract Abstract 2014 Platelet activation by outside-in signaling is initiated by the binding of fibrinogen to alphaIIbbeta3, an integrin only expressed in platelets and megakaryocytes. Signals transduced by alphaIIbbeta3 regulate actin cytoskeleton resulting in filopodia and lamellipodia formation, cell spreading and retraction. c-Cbl protein is abundantly expressed in platelets and functions as E3 ubiquitin ligase and scaffolding protein to mediate protein-protein interactions. Importantly, c-Cbl tyrosine 731 has been shown to interact with p85 subunit of phosphotidylinositol 3-kinase (PI3K) modulating the actin cytoskeleton. Although previous reports showed c-Cbl activation downstream of alphaIIbbeta3, the mechanisms and implications of this activation or the downstream targets remain to be elucidated. We have studied the role of c-Cbl in platelet outside-in signaling: Using human platelets we have demonstrated that c-Cbl Y700, Y731 and Y774 residues undergoes tyrosine phosphorylation upon platelet adhesion to immobilized fibrinogen. These phosphorylation events are completely inhibited in the presence of the pan Src Family Kinases (SFKs) inhibitor (PP2) suggesting that c-Cbl is phosphorylated downstream of SFKs. Spleen tyrosine kinase (Syk) is also involved in this signaling pathway since its inhibition significantly reduce c-Cbl phosphorylation at residues Y774 and Y700; interestingly, tyrosine 731 phosphorylation, which allows the interaction with the p85-subunit of PI3K, is not affected by Syk inhibition. The physiological role of c-Cbl in platelet outside-in signaling was studied using c-Cbl knock-out mice. We found that in contrast to WT platelets, c-Cbl KO platelets had a significantly reduced spreading over a fibrinogen-coated surface. Furthermore, clot retraction analysis demonstrated that c-Cbl KO platelets retraction time was delayed when compared to WT platelets, suggesting a retraction defect. To further elucidate the physiological role of c-Cbl-PI3K interaction we used a knock-in mouse in which the c-Cbl residue Y 731 was substituted with phenylalanine (Y731F) thereby abolishing the PI3K binding site on c-Cbl. Importantly, platelets from Y731F mice showed spreading and clot retraction defect that were comparable with the c-Cbl KO. These result indicates that in large part, the role of c-Cbl in platelets outside-in signaling is determined by its interaction with PI3K. In conclusion, we have demonstrated that c-Cbl plays an important role in platelet outside-in signaling, and its interaction with PI3K through tyrosine 731 is of pivotal importance in platelet spreading and retraction. Disclosures: No relevant conflicts of interest to declare.

PsfR, a factor that stimulates psbAI expression in the cyanobacterium Synechococcus elongatus PCC 7942

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 1031-1040 ◽  
Author(s):  
Colleen Thomas ◽  
Carol R. Andersson ◽  
Shannon R. Canales ◽  
Susan S. Golden
Keyword(s):  
Protein Interactions ◽  
Regulation Of Gene Expression ◽  
D1 Protein ◽  
Direct Interaction ◽  
Synechococcus Elongatus ◽  
Phosphoryl Transfer ◽  
Reaction Centre ◽  
Protein Protein Interactions ◽  
Circadian Expression ◽  
Synechococcus Elongatus Pcc 7942

In this paper a gene (psfR) is reported that regulates psbAI activity in Synechococcus elongatus, a unicellular photoautotrophic cyanobacterium that carries out oxygenic (plant-type) photosynthesis and exhibits global circadian regulation of gene expression. In S. elongatus, a family of three psbA genes encodes the D1 protein of the photosystem II reaction centre. Overexpression of psfR results in increased expression of psbAI, but does not affect the circadian timing of psbAI expression. psfR overexpression affected some, but not all of the genes routinely surveyed for circadian expression. PsfR acts (directly or indirectly) on the psbAI basal promoter region. psfR knockout mutants exhibit wild-type psbAI expression, suggesting that other factors can regulate psbAI expression in the absence of functional PsfR. PsfR contains two receiver-like domains (found in bacterial two-component signal transduction systems), one of which lacks the conserved aspartyl residue required for phosphoryl transfer. PsfR also contains a GGDEF domain. The presence of these domains and the absence of a detectable conserved DNA-binding domain suggest that PsfR may regulate psbAI expression via protein–protein interactions or GGDEF activity (the production of cyclic dinucleotides) rather than direct interaction with the psbAI promoter.

scholarly journals Actin polymerization downstream of integrins: signaling pathways and mechanotransduction

2020 ◽  
Vol 477 (1) ◽  
pp. 1-21 ◽  
Author(s):  
Stéphane Romero ◽  
Christophe Le Clainche ◽  
Alexis M. Gautreau
Keyword(s):  
Actin Cytoskeleton ◽  
Signaling Pathways ◽  
Actin Polymerization ◽  
Cell Attachment ◽  
Direct Interaction ◽  
Directed Migration ◽  
A Cell ◽  
Migration Of Cells ◽  
Intense Research ◽  
Surrounding Extracellular Matrix

A cell constantly adapts to its environment. Cell decisions to survive, to proliferate or to migrate are dictated not only by soluble growth factors, but also through the direct interaction of the cell with the surrounding extracellular matrix (ECM). Integrins and their connections to the actin cytoskeleton are crucial for monitoring cell attachment and the physical properties of the substratum. Cell adhesion dynamics are modulated in complex ways by the polymerization of branched and linear actin arrays, which in turn reinforce ECM-cytoskeleton connection. This review describes the major actin regulators, Ena/VASP proteins, formins and Arp2/3 complexes, in the context of signaling pathways downstream of integrins. We focus on the specific signaling pathways that transduce the rigidity of the substrate and which control durotaxis, i.e. directed migration of cells towards increased ECM rigidity. By doing so, we highlight several recent findings on mechanotransduction and put them into a broad integrative perspective that is the result of decades of intense research on the actin cytoskeleton and its regulation.

scholarly journals The interaction of verprolin WIRE with the adapter proteins family intersectin

2019 ◽  
Vol 17 (1) ◽  
pp. 3-7
Author(s):  
S. V. Kropyvko ◽  
A. V. Rynditch
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Sh3 Domains ◽  
Invasion And Migration ◽  
The Family ◽  
Cellular Signal ◽  
And Migration ◽  
Cytoskeleton Rearrangements

Aim. WIRE is a scafold protein that regulates actin cytoskeleton rearrangements and the formation of actin enriched membrane processes responsible for invasion and migration. ITSN1 and ITSN2 are representatives of the family of intersectins who participate in the reorganization of the actin cytoskeleton, as well as in other processes, such as endo/enzocytosis, cellular signal transduction, etc. As these proteins participate in the same processes, we checked their interaction with each other. Methods. Protein-protein interactions were identified using the GST pull-down method. Results. We showed that the SH3 domains of ITSN1 and ITSN2 interact with WIRE, and found that while WIRE is in a complex with endogenous actin. Conclusions. ITSN1 and ITSN2 interact with WIRE, which is located in a complex with endogenous actin. Keywords: WIRE, ITSN1 and ITSN2, actin.

scholarly journals Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

1999 ◽  
Vol 10 (12) ◽  
pp. 4075-4090 ◽  
Author(s):  
John H. Henson ◽  
Tatyana M. Svitkina ◽  
Andrew R. Burns ◽  
Heather E. Hughes ◽  
Kenneth J. MacPartland ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Sea Urchin ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Wide Spectrum ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Supramolecular Organization ◽  
Cell Periphery ◽  
Retrograde Flow

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

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