Phosphorylation of the LFA-1 Integrin β2-Chain on Thr-758 Leads to Adhesion, Rac-1/Cdc42 Activation, and Stimulation of CD69 Expression in Human T Cells

Nurmi, Susanna M.; Autero, Matti; Raunio, Anna; Gahmberg, Carl G.; Fagerholm, Susanna C.

doi:10.1074/jbc.m608524200

Cited by 52 publications

(67 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: Cell Stimulation Lysis Sds-polyacrylamide Gel Electrophorementioning

confidence: 99%

“…10 Thr758 is a physiologically important amino acid residue in the ␤2 cytoplasmic tail, and becomes phosphorylated after T-cell stimulation with T-cell receptor (TCR) antibodies or with phorbol esters. [11][12][13] After its phosphorylation, ␤2 binds to 14-3-3 proteins both in vitro and in cells. 4 Blocking of this interaction with a ␤2 Thr758 to Ala mutation, or by expression of constructs that bind to 14-3-3 proteins and block their interactions with target proteins, leads to abrogation of actin cytoskeleton rearrangements, cell spreading, and adhesion to ICAM ligands.…”

Section: Introductionmentioning

confidence: 99%

“…4 ␤2-Thr758 phosphorylation leads to the activation of the actin cytoskeleton modulators, Rac1/Cdc42, in cells. 13 The region in the ␤2 cytoplasmic tail that binds 14-3-3 proteins has been reported to interact with filamin in other integrins, 14 and for the strong filamin-binder ␤7 integrin, phosphorylation mimicking substitutions of 3 threonine residues (TTT) reduces filamin affinity. 3 Filamin has been reported to associate with ␤2 integrins in vivo 15,16 and it binds a ␤2 integrin peptide containing the TTT sequence in vitro.…”

Section: Introductionmentioning

confidence: 99%

“…The pThr758-␤2 antibody and the R2E7B antibody to ␤2 have been described previously. 13,17 Predesigned siRNAs for filamin A (siGenome smart pool, M-012579-00) and controls cyclophilin B (siGLO, D-001610-01-05) and nontargeting siRNA (D-001210-02-05) were from Dharmacon (Lafayette, CO). …”

mentioning

confidence: 99%

“…After cell lysis, extracts were run on SDSpolyacrylamide gel electrophoresis (PAGE) and detected with the pThr758 polyclonal antibody as described. 13 Membranes were stripped and reprobed with a ␤2 integrin blotting antibody (R2E7B) to confirm equal loading.For detection of filamin knockdown, 10 6 siRNA-treated cells were centrifuged and the pellet was washed once with PBS. The cells were lysed in NET-buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) including protease inhibitors (Complete; Roche, Indianapolis, IN).…”

mentioning

confidence: 99%

See 4 more Smart Citations

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

Takala

Nurminen

Nurmi³

et al. 2008

Blood

Self Cite

149

235

View full text Add to dashboard Cite

Leukocyte integrins of the ␤2 family are essential for immune cell-cell adhesion. In activated cells, ␤2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the ␤2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of ␤2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated ␤2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K d , 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K d , 0.5 mM). Phosphorylation did not regulate talin binding to ␤2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated ␤2 integrin. X-ray crystallographic data clearly IntroductionIntegrins are heterodimeric plasma membrane receptors that mediate binding to the extracellular matrix and to ligands present on the surface of other cells. Their function is tightly regulated; they bind ligands only after activation. Modulation of integrin activity occurs through tightly regulated interactions between cytoplasmic molecules and integrin intracellular tails. Factors binding to integrin cytoplasmic domains regulating integrin adhesiveness include the cytoskeletal proteins talin 1,2 and filamin, 3 and the 14-3-3 proteins, which are molecular adaptors that bind to phosphorylated serine or threonine (pSer/ pThr) containing polypeptide sequences. 4 The ␤2 integrins are expressed exclusively on leukocytes and bind ICAM molecules on other leukocytes and endothelial cells after cell activation. 5,6 Talin binds to ␤2 integrins in vitro and in cells and is involved in activating the ␤2 integrins, resulting in binding to ICAMs. 1,4,[7][8][9] The ␤2 integrin polypeptide chain is phosphorylated on the intracellular domain on several residues after cell stimulation with various agents. 10 Thr758 is a physiologically important amino acid residue in the ␤2 cytoplasmic tail, and becomes phosphorylated after T-cell stimulation with T-cell receptor (TCR) antibodies or with phorbol esters. [11][12][13] After its phosphorylation, ␤2 binds to 14-3-3 proteins both in vitro and in cells. 4 Blocking of this interaction with a ␤2 Thr758 to Ala mutation, or by expression of constructs that bind to 14-3-3 proteins and block their interactions with target proteins, leads to abrogation of actin cytoskeleton rearrangements, cell spreading, and adhesion to ICAM ligands. 4 ␤2-Thr758 phosphorylation leads to the activation of the actin cytoskeleton modulators, Rac1/Cdc42, in cells. 13 The region in the ␤2 cytoplasmic tail that binds 14-3-3 proteins has been reported to interact with filamin in other integrins, 14 and for the strong filamin-binder ␤7 integrin, phosphorylation mimicking substitutions of 3 threonine residues (TTT) reduces filamin affinity. 3 Fi...

show abstract

Section: Cell Stimulation Lysis Sds-polyacrylamide Gel Electrophorementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

Takala

Nurminen

Nurmi³

et al. 2008

Blood

Self Cite

149

235

View full text Add to dashboard Cite

show abstract

Cysteine protease‐mediated cytoskeleton interactions with LFA‐1 promote T‐cell morphological changes

Jevnikar

Obermajer

Kos

2009

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

T cells migrate through restrictive barriers in a protease-independent, amoeboid fashion that is characterized by morphological cell polarization. The interaction of cysteine-dependent carboxypeptidase cathepsin X with beta(2) integrin LFA-1 (lymphocyte function associated antigen 1) induces T-cell morphological changes, displaying into a 3D extracellular matrix a cytoplasmic projection termed a uropod. In the present study we show that inhibition of cathepsin X and a cysteine-dependent endopeptidase, cathepsin L, markedly inhibits T-cell actin polymerization, shape polarization, and chemotaxis. We propose that cathepsin L promotes T-cell migration associated processes by activating procathepsin X in the endolysosomal vesicles near the cell membrane and at the peak of the uropod, where both proteases were colocalized. We show that active cathepsin X modifies the beta(2) cytoplasmic tail of LFA-1 in the uropod, promoting its high affinity conformation. We suggest that LFA-1 cleavage contributes to the conformational change in the cytoplasmic tail, promoting the binding of the cytoskeletal protein talin. This interaction is restricted to the uropod and results in the stabilization of this region, promoting LFA-1-mediated cell uropod elongation.

show abstract

Cathepsin X cleaves the β2 cytoplasmic tail of LFA‐1 inducing the intermediate affinity form of LFA‐1 and α‐actinin‐1 binding

et al. 2009

View full text Add to dashboard Cite

The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the b 2 chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S 769 , E 768 and A 767 from the C-terminal of the b 2 cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein a-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with a-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration.Key words: a-Actinin-1 . b 2 Cytoplasmic tail . Cathepsin X . LFA-1 affinity . T-cell migration IntroductionIntegrins are a large family of a/b heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix adhesion and transduce signals bi-directionally across the plasma membrane. Lymphocyte function associated antigen-1 (LFA-1, a L b 2 , CD11a/CD18) belongs to the b 2 integrin subfamily and is constitutively expressed on all leukocytes. LFA-1 undergoes a dramatic conformational change that involves affinity regulation and cell surface clustering (valency regulation) during lymphocyte activation, which greatly increases the binding avidity (affinity1valency) for ligands such as ICAM-1, -2 and -3 [1, 2].LFA-1 can take up three conformations, a bent form and two extended forms. The bent form is inactive, whereas the extended forms, closed and open, exhibit intermediate and high affinities, respectively, for ICAM-1, depending on the angle between the hybrid and b-like I domains [3,4]. The extended conformations are recognized by mAb KIM 127 that binds to the epitope on the second EGF-like domain of the b 2 subunit, which is exposed when LFA-1 converts from the bent to the extended form [5,6]. Another antibody, mAb 24, recognizes solely the open, highaffinity conformation of LFA-1 [7].LFA-1 functions either by binding ligands through a process known as outside-in signaling or by binding cytoskeleton components for inside-out signaling. Although the a L and b 2 cytoplasmic tails of LFA-1 are relatively short, they play a pivotal role in these bi-directional signaling processes. For example, truncation of the b 2 cytoplasmic tail eliminates LFA-1 binding to 3 [15] that are the final effectors of inside-out signaling pathways. T-cell migration is a cyclic process that includes alternating integrin-mediated cell adhesion and de-adhesion, associated with regular activation and de-activation of integrins [16,17]. Accumulating...

show abstract

Phosphorylation of the LFA-1 Integrin β2-Chain on Thr-758 Leads to Adhesion, Rac-1/Cdc42 Activation, and Stimulation of CD69 Expression in Human T Cells

Abstract: Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin ␤2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion.

Cited by 52 publications

References 39 publications

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

Cysteine protease‐mediated cytoskeleton interactions with LFA‐1 promote T‐cell morphological changes

Cathepsin X cleaves the β2 cytoplasmic tail of LFA‐1 inducing the intermediate affinity form of LFA‐1 and α‐actinin‐1 binding

Contact Info

Product

Resources

About