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BMC Biology Nov 2020Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by...
BACKGROUND
Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.
RESULTS
We show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.
CONCLUSIONS
We have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.
Topics: Adenosine Triphosphate; Animals; HEK293 Cells; HeLa Cells; Humans; Inflammasomes; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Paxillin; Receptors, Purinergic P2X7
PubMed: 33243234
DOI: 10.1186/s12915-020-00918-w -
Scientific Reports Jul 2019Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial...
Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types from different species the binding dynamics of functionally related FA protein pairs: paxillin and vinculin versus zyxin and VASP. In photobleaching experiments ~40% of paxillin and vinculin remained stably associated with a FA for over half an hour. Zyxin and VASP predominantly displayed more transient interactions. We show protein binding dynamics are influenced by FA location and orientation. In FAs located close to the edge of the adherent membrane paxillin, zyxin and VASP were more dynamic and had larger bound fractions. Zyxin and VASP were also more dynamic and had larger bound fractions at FAs perpendicular compared to parallel to this edge. Finally, we developed a photoconversion assay to specifically visualise stably bound proteins within subcellular structures and organelles. This revealed that while paxillin and vinculin are distributed evenly throughout FAs, their stably bound fractions form small clusters within the FA-complex. These clusters are more concentrated for paxillin than for vinculin and are mostly found at the proximal half of the FA where actin also enters.
Topics: Animals; Bone Neoplasms; Cell Adhesion Molecules; Cytoskeleton; Dogs; Extracellular Matrix; Focal Adhesions; Humans; Madin Darby Canine Kidney Cells; Microfilament Proteins; Osteosarcoma; Paxillin; Phosphoproteins; Tumor Cells, Cultured; Vinculin; Zyxin
PubMed: 31320676
DOI: 10.1038/s41598-019-46905-2 -
HGF/c-Met/β1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells.Life Science Alliance Oct 2023Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and...
Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, β1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and β1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and β1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC.
Topics: Humans; Paxillin; Carcinoma, Non-Small-Cell Lung; Phosphatidylinositol 3-Kinases; Lung Neoplasms; Adenocarcinoma of Lung; Integrins; Hepatocyte Growth Factor
PubMed: 37550007
DOI: 10.26508/lsa.202301953 -
Cellular and Molecular Life Sciences :... Mar 2009The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin...
The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin phosphorylation in normal neutrophils, and that both CXCL8 receptors (CXCR1 and CXCR2) mediated CXCL8-induced paxillin phosphorylation. In CXCR2-transfected cells, the process depended on G(alphai) and G(alphas) coupling to CXCR2. Dominant negative (DN) paxillin increased CXCL8-induced adhesion and migration, indicating that endogenous paxillin keeps migration at submaximal levels. Furthermore, using activating antibodies to beta1 integrins, analyses with focal adhesion kinase (FAK) DN variant (FRNK) and co-immunoprecipitations of FAK and paxillin, we found that beta1 integrin ligation cooperates with CXCL8-induced stimulation, leading to FAK activation and thereafter to FAK-mediated paxillin phosphorylation. Our findings indicate that paxillin keeps directional motility at a restrained magnitude, and suggest that perturbations in its activation may lead to chemotactic imbalance and to pathological conditions associated with excessive or reduced leukocyte migration.
Topics: Animals; Cell Adhesion; Cell Line; Cell Movement; Enzyme Activation; Fibronectins; Focal Adhesion Kinase 2; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Humans; Integrin beta1; Interleukin-8; Neutrophils; Paxillin; Rats; Receptors, Interleukin-8A; Receptors, Interleukin-8B; src-Family Kinases
PubMed: 19151925
DOI: 10.1007/s00018-009-8447-5 -
International Journal of Molecular... Nov 2022Integrins allow cells to adhere to the extracellular matrix and promote the recruitment of other integrins, resulting in the formation of focal adhesion sites at the... (Review)
Review
Integrins allow cells to adhere to the extracellular matrix and promote the recruitment of other integrins, resulting in the formation of focal adhesion sites at the binding sites. Focal adhesion sites play essential roles in the assembly of the cytoskeleton and are vital in shaping the structure of cells. They also play other regulatory roles by influencing numerous biological functions, such as cell proliferation and apoptosis. Hydrogen peroxide‑inducible clone 5 (Hic‑5) is a member of the Paxillin family of proteins and is an adhesive plaque scaffolding protein. Its expression can be detected in both vascular and smooth muscle cells. Thus, it plays an essential role in vascular remodeling, as well as in fibrotic diseases. Hic‑5 functions as a coactivator of steroid receptors, thus playing a role in steroid hormone‑dependent diseases. It also plays a vital role in the invasive metastasis of various types of cancer. Moreover, several studies have demonstrated that Hic‑5 plays a critical role in transcriptional regulation, as well as in numerous signaling pathways. Therefore, the inhibition of the functions of Hic‑5 may prevent the development or halt the progression of several diseases. Its use as a therapeutic target in future investigations may thus aid in the treatment of several diseases, including various types of cancer. The present review article focused on the expression and functions of Hic‑5 in different organs, with the aim of highlighting novel possibilities for future research.
Topics: Cell Adhesion; Focal Adhesion Protein-Tyrosine Kinases; Hormones; Hydrogen Peroxide; Integrins; Paxillin; Phosphorylation
PubMed: 36222304
DOI: 10.3892/ijmm.2022.5194 -
Cell Reports Aug 2022Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in...
Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in extremely soft environments remain unclear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates, growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase. Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates require drebrin phosphorylation and paxillin-drebrin association.
Topics: Actins; Brain-Derived Neurotrophic Factor; Growth Cones; Hydrogels; Neurons; Neuropeptides; Paxillin
PubMed: 35977504
DOI: 10.1016/j.celrep.2022.111188 -
PLoS Computational Biology Jul 2018Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are...
Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are spatiotemporally regulated, with new adhesions forming at the leading edge of the cell and mature adhesions disassembling at the rear. Signalling proteins and structural cytoskeletal components tightly regulate adhesion dynamics. Paxillin, an adaptor protein within adhesions, is one of these proteins. Its phosphorylation at serine 273 (S273) is crucial for maintaining fast adhesion assembly and disassembly. Paxillin is known to bind to a GIT1-βPIX-PAK1 complex, which increases the local activation of the small GTPase Rac. To understand quantitatively the behaviour of this system and how it relates to adhesion assembly/disassembly, we developed a mathematical model describing the dynamics of the small GTPases Rac and Rho as determined by paxillin S273 phosphorylation. Our model revealed that the system possesses bistability, where switching between uninduced (active Rho) and induced (active Rac) states can occur through a change in rate of paxillin phosphorylation or PAK1 activation. The bistable switch is characterized by the presence of memory, minimal change in the levels of active Rac and Rho within the induced and uninduced states, respectively, and the limited regime of monostability associated with the uninduced state. These results were validated experimentally by showing the presence of bimodality in adhesion assembly and disassembly rates, and demonstrating that Rac activity increases after treating Chinese Hamster Ovary cells with okadaic acid (a paxillin phosphatase inhibitor), followed by a modest recovery after 20 min washout. Spatial gradients of phosphorylated paxillin in a reaction-diffusion model gave rise to distinct regions of Rac and Rho activities, resembling polarization of a cell into front and rear. Perturbing several parameters of the model also revealed important insights into how signalling components upstream and downstream of paxillin phosphorylation affect dynamics.
Topics: Animals; CHO Cells; Cell Movement; Cell Polarity; Cricetulus; Cytoskeletal Proteins; Enzyme Activation; Enzyme Inhibitors; Focal Adhesions; Models, Biological; Okadaic Acid; Paxillin; Phosphorylation; Serine; Signal Transduction; p21-Activated Kinases; rac GTP-Binding Proteins; rho GTP-Binding Proteins
PubMed: 29975690
DOI: 10.1371/journal.pcbi.1006303 -
Molecular Biology of the Cell Dec 2019Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical...
Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical states, such as cell shape and spreading area. Many of these responses are believed to involve coupled output and input of mechanical forces, which may constitute the key sensing mechanism to generate downstream regulatory signals for cell growth and differentiation. Here, we show that the state of cell migration also plays a regulatory role. Compared with migrating cells, stationary cells generate stronger, less dynamic, and more peripherally localized traction forces. These changes are coupled to reduced focal adhesion turnover and enhanced paxillin phosphorylation. Further, using cells migrating along checkerboard micropatterns, we show that the appearance of new focal adhesions directly in front of existing focal adhesions is associated with the down-regulation of existing focal adhesions and associated traction forces. Together, our results imply a mechanism where cell migration regulates traction forces by promoting dynamic turnover of focal adhesions, which may then regulate processes such as wound healing and embryogenesis where cell differentiation must coordinate with migration state and proper localization.
Topics: 3T3 Cells; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Line; Cell Movement; Cell Shape; Focal Adhesions; Mice; Paxillin; Phosphorylation
PubMed: 31693433
DOI: 10.1091/mbc.E19-02-0099 -
Journal of Cell Science Aug 2008Paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of sites of cell adhesion to the extracellular matrix. Through the interactions... (Review)
Review
Paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of sites of cell adhesion to the extracellular matrix. Through the interactions of its multiple protein-binding modules, many of which are regulated by phosphorylation, paxillin serves as a platform for the recruitment of numerous regulatory and structural proteins that together control the dynamic changes in cell adhesion, cytoskeletal reorganization and gene expression that are necessary for cell migration and survival. In particular, paxillin plays a central role in coordinating the spatial and temporal action of the Rho family of small GTPases, which regulate the actin cytoskeleton, by recruiting an array of GTPase activator, suppressor and effector proteins to cell adhesions. When paxillin was first described 18 years ago, the amazing complexity of cell-adhesion organization, dynamics and signaling was yet to be realized. Herein we highlight our current understanding of how the multiple protein interactions of paxillin contribute to the coordination of cell-adhesion function.
Topics: Animals; Cell Adhesion; Cell Movement; Cell Survival; Cytoskeleton; Humans; Models, Biological; Paxillin; Phosphorylation; rho GTP-Binding Proteins
PubMed: 18650496
DOI: 10.1242/jcs.018044 -
Biomolecules Jul 2022SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly...
SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.
Topics: Nuclear Proteins; Oncogenes; Paxillin; Phosphorylation; Protein Binding; Tyrosine; src Homology Domains
PubMed: 35892324
DOI: 10.3390/biom12081014