β1 Integrin-dependent binding of Jurkat cells to fibronectin is regulated by a serine-threonine phosphatase

Abstract

We investigated the effects of signaling molecule inhibitors on the expression and function of ␤ 1 integrins in Jurkat cells. Jurkat cells expressed ␣ 4 ␤ 1 and ␣ 5 ␤ 1 , with significant levels of constitutively activated ␤ 1 integrins as assessed by labeling with mAb 15/7 that distinguishes between activation states. Adhesion to fibronectin (Fn) was mediated equally through ␣ 4 and ␣ 5 subunits, and was potentiated by the ␤ 1 integrin activating mAb 8A2. Fn adhesion was decreased by okadaic acid through effects on both ␣ 4 ␤ 1 and ␣ 5 ␤ 1 . Tyrphostin A23 also decreased adhesion but was less potent. Neither inhibitor had any effect on the surface expression of total or activated ␤ 1 integrins. The effect of tyrphostin was completely reversed by 8A2; the effect of okadaic acid was only partially reversed. Using Calyculin A, we determined that Jurkat adhesion to Fn was regulated via protein phosphatase 1, independent of the levels of integrins or integrin activation epitopes. Activation of Jurkat cells with a CD3-stimulating mAb enhanced adhesion to Fn and was partially blocked by okadaic acid. These data demonstrate different regulatory pathways for constitutive versus activationdependent adhesion via ␤ 1 integrins, and implicate both tyrosine kinases and serine-threonine phosphatases in integrin function. J. Leukoc. Biol. 64: 753-758; 1998.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

“…Jurkat cells were chosen for studying integrin β 1 interactions, as this human T cell line expresses high amounts of this integrin and has been used extensively for its investigation before [25]. Different concentrations of formaldehyde (0.4% to 2%) were used to cross-link Jurkat cells.…”

“…Furthermore we consistently identified integrin α 4. The receptor formed by these integrins is VLA-4 (very late antigen 4), a known receptor found on T cells [31] and a high abundant integrin on Jurkat cells [25]. …”

“…The listed proteins were not detected in control experiments using either non-formaldehyde treated cells or immunoprecipitations performed without the antibody when analysing gel bands at masses corresponding to the integrin β 1 containing complex. Of these additionally identified proteins, integrin α 6 is known to form a receptor termed VLA-6 together with integrin β 1 on T cells, which has not been detected in Jurkat cells before [25]. Two additional integrin α subunits were detected only in dataset 2: integrin α 5 (log(e) = −36.4) and α 1 (log(e) = −1.7).…”

“…Two additional integrin α subunits were detected only in dataset 2: integrin α 5 (log(e) = −36.4) and α 1 (log(e) = −1.7). Integrin α 5 had been demonstrated to be expressed together with integrin β 1 on Jurkat cells [25], whereas integrin α 1 is known to form a collagen-recognizing receptor with integrin β 1 on endothelial cells [32]. Even though only few peptides of each protein were sequenced, the identification of the integrin α subunits is reliable as they do not share high homologues sequences and all sequenced peptides were unique for each integrin α .…”

Cadherin 26 is an alpha integrin-binding epithelial receptor regulated during allergic inflammation

“…Jurkat cells, which express integrin α4β1 but not α4β7 35 , adhered to CDH26-Fc to a significantly greater degree than they adhered to IgG control antibody (Figure 4i). Pre-incubation of Jurkat cells with anti-integrin α4 antibody (HP2/1), but not an equivalent amount of control mIgG1, blocked their binding to CDH26-Fc in a dose-dependent manner (Figure 4i).…”