The kindlin 3 pleckstrin homology domain has an essential role in lymphocyte function-associated antigen 1 (LFA-1) integrin-mediated B cell adhesion and migration

Abstract

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.

Keywords: Adhesion; Cell Migration; Cell Signaling; Integrin; Kindlin; Lymphocyte; PH Domain; Receptors.

FIGURE 1.

Diagrammatic representation of kindlin 3 PH domain and kindlin 3-GFP constructs showing the FERM subdomains (residues 1–663). A, model of the kindlin 3 PH domain showing details of the β1-β2 loop and location of the IPRR insert at the junction of the β1 strand and β1-β2 loop. Inset, expansion of the β1-β2 loop indicating the amino acids that comprise the loop and illustrating the predominance of positively charged residues with IP₄ interactive residues as in kindlin 2 highlighted in red (30). B, amino acid sequence covering the β1 (β1.1 and β1.2) and β2 strands and major phosphoinositide binding β1-β2 loop (IP₄ binding residues, red). C, details of the human kindlin 3-GFP chimeric proteins that are used in the study. The wild type kindlin 3 protein contains an F0 domain, F1 domain, F2 domain intersected by a PH domain, and an F3 domain that binds to the β subunit of integrin (kindlin 3-GFP). Also illustrated are a second kindlin 3 splice variant with residues IPRR inserted into the PH domain (yellow line) (kindlin 3-IPRR-GFP), kindlin 3 with the PH domain deleted (kindlin 3-ΔPH-GFP), and the F2 subdomain that contains the PH domain (kindlin 3-PH-GFP).

FIGURE 2.

Phosphoinositide binding properties of the kindlin 3-GFP proteins. A, binding of wild type kindlin 3-GFP to control-, IP₃-, and IP₄-coupled beads revealed by blotting and anti-GFP mAb. The blot is representative of three independent experiments. B, comparison of control and IP₄ bead binding properties of wild type kindlin 3-GFP and isolated FERM subdomain 2 incorporating the PH domain (kindlin 3 PH-GFP). Input, amount of GFP protein added to beads; output, amount of GFP protein bound to beads following incubation and washing. C, quantification of kindlin 3-GFP and kindlin 3 PH-GFP binding to control and IP₄ beads from three independent experiments. Density was normalized to that of the relevant input band and binding to control beads. Data are shown as mean ± S.E. (error bars); NS, not significant. D, comparison of IP₃ and IP₄ bead binding of wild type kindlin 3-GFP, kindlin 3-ΔPH-GFP, and kindlin 3-IPRR-GFP. Input and output are as in B. Blot is representative of three independent experiments. E, quantification of kindlin 3-GFP, kindlin 3-ΔPH-GFP, and kindlin 3-IPRR-GFP binding to IP₃ and IP₄ beads from three independent experiments. Density was normalized to that of relevant input band and kindlin 3 binding to IP₃ beads. Data are shown as mean ± S.E.; *, p < 0.05; NS, not significant.

FIGURE 3.

Adhesion characteristics of LAD-III B cells expressing human wild type or mutant kindlin 3-GFP. A, phase and IRM images of a LAD-III patient's EBV-transformed B cells transfected with eGFP cDNA compared with cells transfected with wild type KINDLIN3, KINDLIN3-IPRR, and KINDLIN3-ΔPH cDNAs. These cells were compared with parent B cells that expressed kindlin 3 endogenously and were transfected with eGFP cDNA (control). Images are representative of 20 fields/sample; n = 3; scale bar, 5 μm. B, quantification of the area of close contact from three independent experiments (>60 cells/group). Data are shown as mean ± S.E. (error bars); ***, p < 0.001; NS, not significant. C, expression levels of kindlin 3-GFP and GFP proteins in LAD-III EBV-transformed B cells. Gating shows that the KINDLIN3-GFP cDNAs (10 μg/2 × 10⁷ cells) and eGFP cDNA (2 μg/2 × 10⁷ cells) are translated in EBV-transformed B cells with a comparable efficiency at 24 h. D, typical experiment showing equal expression levels of GFP, kindlin 3-GFP, kindlin 3-ΔPH-GFP, and kindlin 3-IPRR-GFP proteins in transfected B cells compared with mock-transfected cells as determined by flow cytometry.

FIGURE 4.

Migration characteristics of LAD-III B cells expressing human wild type or mutant kindlin 3-GFP. A, representative single cell tracking images showing migration characteristics of control B cells and LAD-III B cells transfected with eGFP cDNA compared with cells transfected with wild type KINDLIN3, KINDLIN3-IPRR, and KINDLIN3-ΔPH cDNAs; n = 3 for each cDNA construct. B, quantification of average speed of migration for n = 15 cells/group from a representative experiment. Data are shown as mean ± S.E. (error bars); ***, p < 0.001; NS, not significant.

FIGURE 5.

TIRF analysis of LAD-III B cells attached to ICAM-1 and poly-l-lysine. A, phase and TIRF images at the plasma membrane of LAD III B cells attached to ICAM-1. Cells transfected with eGFP cDNA were compared with cells transfected with wild type KINDLIN3, KINDLIN3-IPRR, and KINDLIN3-ΔPH cDNAs. Images are representative of 20 fields/sample; n = 4; scale bar, = 5 μm. B, quantification of the mean fluorescence intensity of GFP at the cell membrane level with ICAM-1, normalized to that of the LAD-III B cells transfected with eGFP cDNA. Data are shown as mean ± S.E. (error bars) from four independent experiments (>95 cells/group); *, p < 0.05; ***, p < 0.01; ****, p < 0.0001; NS, not significant. C, phase and TIRF images of the plasma membrane of LAD-III B cells at the interface with poly-l-lysine. Cells transfected with eGFP cDNA were compared with cells transfected with wild type KINDLIN3, KINDLIN3-IPRR, and KINDLIN3-ΔPH cDNAs taken at the level of the cell membrane with poly-l-lysine. Images are representative of 20 fields/sample; n = 4; scale bar, 5 μm. D, quantification of the mean fluorescence intensity of GFP at the cell membrane level with poly-l-lysine, normalized to that of the LAD-III B cells transfected with eGFP cDNA. Data are shown as mean ± S.E. from four independent experiments (>95 cells/group); *, p < 0.05; **, p < 0.01; ****, p < 0.0001; NS, not significant. E, left, quantification of the mean fluorescence intensity of GFP at the cell membrane level with ICAM-1, normalized to that of the LAD-III B cells transfected with eGFP cDNA. E, right, quantification of the area of cell membrane LFA-1 at the interface with ICAM-1. Cells were treated ± 5 μm LY294002 for 1 h. Data are shown as mean ± S.E. from three independent experiments (>60 cells/group); *, p < 0.05; ****, p < 0.0001; NS, not significant.