scholarly journals New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

2022 ◽  
Vol 5 (4) ◽  
pp. e202101301
Author(s):  
Ralph T Böttcher ◽  
Nico Strohmeyer ◽  
Jonas Aretz ◽  
Reinhard Fässler
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Phosphorylation Site ◽  
Cell Spreading ◽  
Mouse Cell ◽  
Β1 Integrin ◽  
Β1 Integrins ◽  
Integrin Ligand ◽  
Human And Mouse ◽  
Major Phosphorylation Site

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

scholarly journals 3-O-Deacylation of Lipid A by PagL, a PhoP/PhoQ-regulated Deacylase ofSalmonella typhimurium, Modulates Signaling through Toll-like Receptor 4

2004 ◽  
Vol 279 (19) ◽  
pp. 20044-20048 ◽  
Author(s):  
Kiyoshi Kawasaki ◽  
Robert K. Ernst ◽  
Samuel I. Miller
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Host Defense ◽  
Lipid A ◽  
Virulence Gene ◽  
Mouse Cell ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Layer Chromatography ◽  
Human And Mouse

Toll-like receptor 4 (TLR4)-mediated responses, which are induced by the lipid A portion of lipopolysaccharide, are important for host defense against Salmonellae infection. A variety of different data indicate that the acylation state of lipid A can alter TLR4-mediated responses. TheS. typhimuriumvirulence gene product PhoP/PhoQ signals the presence of host microenvironments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. We now demonstrate that 3-O-deacylation and palmitoylation of lipid A decreases its ability to induce TLR4-mediated signaling. Deacylated lipid A, deacylated and palmitoylated lipid A, palmitoylated lipid A, and unmodified lipid A species were purified fromEscherichia coliheterologously expressing PagL and/or PagP. The purified lipid A preparations showed spectra of a single lipid A species on mass spectrometry and gave a single band on thin layer chromatography. The activity of purified lipid A species was examined using human and mouse cell lines that express recombinant human TLR4. Compared with unmodified lipid A, the modified lipid A species are 30–100-fold less active in the ability to induce NF-κB-dependent reporter activation. These results suggest that the lipid A modifications reduce TLR4-signaling as part of Salmonellae adaptation to host environments.

scholarly journals AMPK negatively regulates tensin-dependent integrin activity

2017 ◽  
Vol 216 (4) ◽  
pp. 1107-1121 ◽  
Author(s):  
Maria Georgiadou ◽  
Johanna Lilja ◽  
Guillaume Jacquemet ◽  
Camilo Guzmán ◽  
Maria Rafaeva ◽  
...  
Keyword(s):  
Adhesion Formation ◽  
Cell Spreading ◽  
Fiber Formation ◽  
Β1 Integrin ◽  
Cellular Functions ◽  
Cell Matrix ◽  
Cytoplasmic Tails ◽  
Integrin Ligand ◽  
Fibrillar Adhesion ◽  
Amp Activated Protein Kinase

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin–ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin– and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

scholarly journals Reovirus directly engages integrin to recruit clathrin for entry into host cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Melanie Koehler ◽  
Simon J. L. Petitjean ◽  
Jinsung Yang ◽  
Pavithra Aravamudhan ◽  
Xayathed Somoulay ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Entry ◽  
Host Factors ◽  
Host Cells ◽  
Viral Gene ◽  
Β1 Integrin ◽  
Force Microscopy ◽  
Β1 Integrins ◽  
Integrin Ligand ◽  
Viral Gene Delivery

AbstractReovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

scholarly journals Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Marina Theodosiou ◽  
Moritz Widmaier ◽  
Ralph T Böttcher ◽  
Emanuel Rognoni ◽  
Maik Veelders ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Hematopoietic Cells ◽  
Cell Spreading ◽  
Integrin Activation ◽  
Independent Manner ◽  
High Affinity ◽  
Adhesion Sites ◽  
Β1 Integrins

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.

Differences in phosphatase modulation of α4 β1 and α5 β1 integrin-mediated adhesion and migration of B16F1 cells

1999 ◽  
Vol 77 (5) ◽  
pp. 409-420 ◽  
Author(s):  
Dolores Hangan-Steinman ◽  
Wai-chi Ho ◽  
Priti Shenoy ◽  
Bosco MC Chan ◽  
Vincent L Morris
Keyword(s):  
Cell Adhesion ◽  
Adhesive Strength ◽  
Cell Movement ◽  
Protein Tyrosine Phosphatases ◽  
Β1 Integrin ◽  
Phosphatase Inhibitors ◽  
Β1 Integrins ◽  
Cell Adhesion And Migration ◽  
And Migration ◽  
B16f1 Cell

It is well established that a biphasic relationship exists between the adhesive strength of β1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in β1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in α4β1 and α5β1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, α5β1 functioned as the predominant receptor for cell movement; a role for α4β1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of α4β1 and α5β1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of α5β1, but not α4β1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of β1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various β1 integrins exist. Key words: phosphatases, integrins, cell movement, cell adhesion.

scholarly journals Yersinia enterocolitica Adhesin A Induces Production of Interleukin-8 in Epithelial Cells

2004 ◽  
Vol 72 (12) ◽  
pp. 6780-6789 ◽  
Author(s):  
Yvonne Schmid ◽  
Guntram A. Grassl ◽  
Oliver T. Bühler ◽  
Mikael Skurnik ◽  
Ingo B. Autenrieth ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Yersinia Enterocolitica ◽  
Hela Cells ◽  
Map Kinases ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Interleukin 8 ◽  
Β1 Integrin ◽  
Β1 Integrins ◽  
Tissue Reactions

ABSTRACT The major invasive factor of Yersinia enterocolitica, the invasin (Inv) protein, induces proinflammatory host cell responses, including interleukin-8 (IL-8) secretion from human epithelial cells, by engagement of β1 integrins. The Inv-triggered β1 integrin signaling involves the small GTPase Rac; the activation of MAP kinases, such as p38, MEK1, and JNK; and the activation of the transcription factor NF-κB. In the present study, we demonstrate that Y. enterocolitica YadA, which is a major adhesin of Y. enterocolitica with pleiotropic virulence effects, induces IL-8 secretion in epithelial cells. The abilites of YadA and Inv to promote adhesion to and invasion of HeLa cells and to induce IL-8 production by the cells were investigated by expression of YadA and Inv in Escherichia coli. While YadA mediates efficacious adhesion to HeLa cells, it mediates marginal invasion compared with Inv. Both YadA and Inv trigger comparable levels of IL-8 production. Conformational changes of the YadA head domain by mutation of NSVAIG-S motifs, which abolish collagen binding, also abolish adhesion of Yersinia to HeLa cells and YadA-mediated IL-8 secretion. Furthermore, experiments in which blocking antibodies against β1 integrins were used demonstrate that β1 integrins are crucial for YadA-mediated IL-8 secretion. Inhibitor studies demonstrate the involvement of small GTPases and MAP kinases, such as p38, MEK1, and JNK, indicating that β1 integrin-dependent signaling mediated by Inv or YadA involves similar signaling pathways. These data present YadA, in addition to Inv, YopB, and Yersinia lipopolysaccharide, as a further inducer of proinflammatory molecules by which Y. enterocolitica might promote inflammatory tissue reactions.

Differential Integrin Expression Facilitates Isolation of Human Fetal Pancreatic Epithelial Cells

1994 ◽  
Vol 3 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Fred Levine ◽  
Gillian M. Beattie ◽  
Alberto Hayek
Keyword(s):  
Epithelial Cells ◽  
Stromal Cells ◽  
Immunohistochemical Staining ◽  
Endocrine Cells ◽  
Yersinia Pseudotuberculosis ◽  
Affinity Binding ◽  
Β1 Integrin ◽  
Human Pancreas ◽  
High Affinity ◽  
Β1 Integrins

We have studied the expression of the β1 family of integrins in fetal and adult human pancreas. Immunohistochemical staining with a monoclonal anti-β1 antibody revealed that the epithelial cells of the human fetal pancreas express high amounts of β1 integrin, while the pancreatic stromal cells express substantially lower amounts. Islets of Langerhans from human adult pancreas also expressed high amounts of β1 integrin. Taking advantage of the extremely high affinity binding between the invasin protein of Yersinia pseudotuberculosis and many β1 integrins, we have been able to isolate highly enriched populations of fetal pancreatic epithelial cells. Epithelial-enriched cell populations retain the ability to differentiate into mature endocrine cells following transplantation into nude mice.

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