Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization

Abstract

The guanine nucleotide exchange factor Vav1 regulates actin polymerization and contributes to cytotoxicity by natural killer (NK) cells. An open question is how Vav1 becomes activated and what receptor can signal upstream of actin cytoskeleton rearrangement upon NK cell contact with target cells. Using transfected insect cells that express ligands of human NK cell receptors, we show that engagement of the beta2 integrin LFA-1 on NK cells by intercellular adhesion molecule (ICAM)-1 led to a tyrosine phosphorylation of Vav1 that was not sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 phosphorylation was blocked by an inhibitor of Src-family kinases, and correlated with activation of its downstream effector PAK. Binding of activation receptor 2B4 to its ligand CD48 was not sufficient for Vav1 phosphorylation. However, coengagement of 2B4 with LFA-1 resulted in an enhancement of Vav1 phosphorylation that was sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 was recruited to a detergent-resistant membrane (DRM) fraction only when 2B4 and LFA-1 were coengaged, but not after LFA-1 engagement. Therefore, binding of LFA-1 to ICAM-1 on target cells may initiate an early signaling cascade in NK cells through activation of Vav1, leading to cytoskeleton reorganization and amplification of signals from other activation receptors.

Figure 1.

Tyrosine phosphorylation of Vav1 after antibody-induced engagement of LFA-1 in NK cells. LFA-1 on NK cells was cross-linked with anti-CD11a and a F(ab′)₂ goat anti–mouse antibody for the indicated times at 37°C. (A) Cell lysates were resolved by 4–20% SDS-PAGE and immunoblotted with biotinylated anti phosphotyrosine Ab, PY-20 (p-Tyr, left). (B) Vav1 was immunoprecipitated from cell lysates, separated on 6% SDS-PAGE, and blotted with PY-20 mAb (p-Tyr, left). The same blots were reprobed with an anti-Vav1 Ab (Vav1, right).

Figure 2.

Phosphorylation of Vav1 induced by engagement of LFA-1 on NK cells. (A) NK cells, pretreated with anti-CD11b or anti-CD11a, were mixed with SC2 or SC2-ICAM-1 target cells. Vav1 was immunoprecipitated from cell lysates, resolved on a 6% SDS-PAGE, and blotted with biotinylated anti phosphotyrosine Ab PY-20 (p-Tyr). The same blot was reblotted with an anti-Vav1 Ab (Vav1). (B) PAK kinase activity in NK cells mixed with SC2 and SC2-ICAM cells. PAK or control (rabbit IgG) immunoprecipitates were subjected to an in vitro kinase assay. Phosphorylation of the MBP substrate (p-MBP) was detected by autoradiography. Vav1 was immunoprecipitated from the supernatant of the PAK immunoprecipitation, and tested for tyrosine phosphorylation as in (A). (C) Inhibition of Vav1 tyrosine phosphorylation. Vav1 phosphorylation was tested in NK cells either untreated (−), treated with PP1, or treated with Piceatannol (Pic), after mixing with SC2 and SC2-ICAM cells, as indicated.

Figure 3.

Phosphorylation of Vav1 induced by engagement of LFA-1 and of 2B4 on NK cells. (A) NK cells were mixed with SC2 (−), SC2-CD48 (CD48), SC2-ICAM-1 (ICAM), or SC2-ICAM-CD48 (ICAM+CD48) target cells. Vav1 was immunoprecipitated, resolved on a 6% SDS-PAGE, and blotted with biotinylated-PY-20 (p-Tyr, left). The same blot was reblotted with an anti-Vav1 Ab (Vav1). (B and C) Vav1 phosphorylation induced by LFA-1 is independent of actin polymerization and of lipid rafts. NK cells treated with cytochalasin D (Cyt.D) and Latrunculin A (B), or with MCD (C) were mixed with SC2 (−), SC2-ICAM (ICAM), or SC2-ICAM-CD48 (ICAM+CD48) cells. Vav1 phosphorylation was detected as in (A).

Figure 4.

Vav1 phosphorylation and translocation into DRM fractions. (A) DRM domains of polyclonal NK cells were characterized by Western blot analysis. Blots were probed with cholera toxin B subunit (CtxB) and anti-CD45 Ab (CD45). Vav1 immunoprecipitated from gradient fractions 2–12 was Western blotted with anti-Vav1 Ab (Vav1). (B) NK cells that were pretreated with (+) or without (−) cytochalasin D (Cyt D), were mixed with SC2 (−), SC2-ICAM (ICAM), or SC2- ICAM-CD48 (ICAM+CD48) cells. Cells were lysed, and DRM isolated using sucrose gradient ultracentrifugation. Fractions 3, 4, and 5 were pooled and correspond to the DRM fractions (DRM), whereas pooled fractions 10, 11, and 12 correspond to the soluble fraction (soluble). Lysates of each pool were analyzed by Western blot with cholera toxin B subunit (CtxB) and anti-CD45 (CD45) Ab. Immunoprecipitated Vav1 was detected by Western blot with anti Vav1 Ab (Vav1). Phospho-Vav1 was detected by blotting the same membrane with an anti-phosphotyrosine Ab, PY-20 (p-Tyr).