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The Journal of Immunology, 2003, 170: 5912-5918.
Copyright © 2003 by The American Association of Immunologists

Paxillin Binding to the {alpha}4 Integrin Subunit Stimulates LFA-1 (Integrin {alpha}L{beta}2)-Dependent T Cell Migration by Augmenting the Activation of Focal Adhesion Kinase/Proline-Rich Tyrosine Kinase-21

David M. Rose2,3,*, Shouchun Liu2,{dagger}, Darren G. Woodside{dagger}, Jaewon Han{dagger}, David D. Schlaepfer{ddagger} and Mark H. Ginsberg{dagger}

* Division of Rheumatology, Allergy, and Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093; and {dagger} Division of Vascular Biology, Department of Cell Biology, and {ddagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Engagement of very late Ag-4 (integrin {alpha}4{beta}1) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin {alpha}L{beta}2). This form of integrin trans-regulation in T cells requires the binding of paxillin to the {alpha}4 integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an {alpha}4 mutation ({alpha}4(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an {alpha}4-binding fragment of paxillin that blocks the {alpha}4-paxillin interaction, selectively blocked VCAM-1 stimulation of {alpha}L{beta}2-dependent cell migration. The {alpha}4-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the {alpha}4 tail resulted in much less {alpha}4{beta}1-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked {alpha}4{beta}1 stimulation of {alpha}L{beta}2-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin {alpha}L{beta}2 by {alpha}4{beta}1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The migration of leukocytes from the vasculature is essential to the development of the immune system, to leukocyte recirculation, and to the control of the inflammatory response (1). These emigration decisions are controlled by multiple adhesion receptors including integrins (1, 2, 3, 4, 5). Integrins are involved in leukocyte trafficking both during immune system development and surveillance and during inflammation. Integrins also transmit cellular signals, which regulate functions such as leukocyte growth, development, and activation. Signals initiated by engagement of one integrin can alter the function of a second integrin, a process termed integrin trans-regulation or cross-talk (6, 7, 8, 9, 10, 11, 12, 13, 14). A form of trans-regulation relevant to the immune response occurs between two integrins essential for leukocyte emigration, integrin {alpha}4{beta}1 (very late Ag-4 (VLA-4)4) and integrin {alpha}L{beta}2 (LFA-1). In particular, engagement of {alpha}4{beta}1 stimulates {alpha}L{beta}2-dependent cell adhesion and migration (6, 7, 8, 9). The trans-regulation between these two integrins may then coordinate leukocyte movement from blood into peripheral tissues.

Integrin signaling often depends on binding of cytoplasmic proteins to integrin cytoplasmic domains (tails) (10). Multiple cellular signaling pathway(s) have been implicated in different forms of integrin trans-regulation (11, 12, 13, 14). This signaling ultimately leads to changes in integrin activation (affinity and/or avidity), which can affect cell adhesion and migration (11, 12, 13, 14). However, the most proximal events in such trans-regulation, i.e., the proteins that bind to the cytoplasmic tails that lead to the signaling events, have remained obscure. {alpha}4 integrins bind tightly to the signaling adaptor molecule, paxillin (15, 16). The high affinity of the {alpha}4 cytoplasmic domain for paxillin leads to distinct cellular responses to ligation of integrin {alpha}4{beta}1. Paxillin functions as a signaling adapter molecule and can associate with nonreceptor tyrosine kinases such as focal adhesion kinase (FAK) and its paralog, proline-rich tyrosine kinase-2 (Pyk2) (17, 18, 19). Both FAK and Pyk2 have been implicated in regulating cell migration (20, 21, 22). In the current work, we analyzed the role of the paxillin-{alpha}4 interaction in integrin trans-regulation by genetic reconstitution of {alpha}4 null Jurkat T cells with mutant {alpha}4 that lacks the paxillin-binding function. Here we report that the integrity of the {alpha}4 paxillin-binding site is required for its capacity to regulate {alpha}L{beta}2-dependent migration. Furthermore, transfection of Jurkat cells with a novel dominant negative paxillin construct selectively perturbed {alpha}4-initiated trans-regulation but had little effect on direct {alpha}L{beta}2-mediated cell migration. In addition, we found that the {alpha}4-paxillin interaction leads to enhanced activation of the related tyrosine kinases Pyk2 and FAK and that inhibition of these kinases abolished trans-regulation. Thus, we define a proximal protein-protein interaction required for trans-regulation between integrins {alpha}4{beta}1 and {alpha}L{beta}2 and identify activation of Pyk2 and FAK as essential signaling events in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The Jurkat E6-1 T leukemic cell line was purchased from American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (BioWhittaker), 1% glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO).

Reagents

The anti-human {beta}1 mAb, 8A2, was the generous gift of Drs. N. Kovach and J. Harlan (University of Washington, Seattle, WA). The anti-human {alpha}4, HP2/1, anti-human {alpha}5, SAM1, and anti-human {beta}1, K20 Abs were purchased from Immunotech (Westbrook, ME). The anti-human {beta}2 mAb hybridoma cell line, TS1/18, was obtained from ATCC and was used to generate ascites fluid. The cDNA encoding the CS-1 region of fibronectin fused to GST was a gift from Dr. J. W. Smith (Burnham Institute, La Jolla, CA). The expression and purification of this fusion protein have been previously described (23). VCAM-Ig consisting of the complete seven Ig domains of the extracellular region of VCAM-1 fused to the heavy chain of human IgG1 and ICAM-Ig consisting of the first two N-terminal Ig domains of ICAM-1 fused to the heavy chain of human IgG1 were expressed and purified as previously described (8). cDNAs for HA-tagged FAK-related nonkinase (FRNK), and FRNK-L/S (L1034S substitution) in pcDNA3.1 have been previously described (24, 25). Myc-tagged Pyk2-hemagglutinin (HA) nonkinase (PRNK) was constructed as follows. PCR was used to amplify the human Pyk2 C-terminal domain using the primers (sense) 5'-AAAAGGATCCATGGAGAAGGACATTGCCATGG-3' and (antisense) 5'-TTTTTCTAGACGCAGGCAGGTGG-3'. The 1016-bp product was digested with BamHI and XbaI and cloned into BglII and XbaI sites of pCS-Myc-tag (24). The 6x-Myc-tag-PRNK construct was removed from pCS by BamHI and XbaI digestion and cloned into the same sites of pcDN3.1. The Myc-tagged fragments of paxillin-encoding regions A176–L277 (pax176–277) and M1–L125 (pax1–125) in pcDAN3.1 were used as described (26).

Generation of {alpha}4-deficient Jurkat cells and their reconstitution with {alpha}4 integrin

Jurkat cells were treated with ethyl methanesulfonate (200 µg/ml; Sigma-Aldrich) for 24 h. After 5 days in culture, cells were stained for {alpha}4 with mAb HP2/1 and low Ab-binding cells were isolated by cell sorting using a FACStarPlus flow cytometer (BD Biosciences, San Jose, CA) into 96-well tissue culture-treated plates (Costar; Corning, Corning, NY). Isolated clonal lines were sequentially reassayed for lack of {alpha}4 expression by flow cytometry with mAb HP2/1 as well as anti-{alpha}4 Ab 9F10. Deficiency in {alpha}4 expression were also verified by immunoblotting. No {alpha}4 protein was detected in whole cell lysates.

Wild-type {alpha}4 ({alpha}4Wt) or {alpha}4 with an alanine substitution at tyrosine 991({alpha}4Y991) was reintroduced back into the {alpha}4-deficient cell line. cDNAs encoding {alpha}4Wt or {alpha}4(Y991A) in pcDNA3.1 were transfected into cells by electroporation as previously described (15). Cells were grown in G418 (1 mg/ml) for 3 wk. Afterward, cells were stained for {alpha}4 with mAb HP2/1 and FACS sorted as described above. Clonal lines were reassayed for {alpha}4 expression by flow cytometry and immunoblotting. Lines expressing equivalent {alpha}4{beta}1 as well as {beta}2 integrin were used for subsequent analysis.

Soluble VCAM-Ig binding assay

Cells (5 x 105) were resuspended in modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 1 mg/ml glucose, and 1 mg/ml BSA) containing 1 mM CaCl2 and 1 mM MgCl2. The VCAM-Ig fusion protein was added at a final concentration of 100 nM and incubated for 30 min at room temperature. Cells were washed twice in Tyrode’s buffer and resuspended in the same buffer containing FITC-conjugated donkey anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1/100 dilution. After 30 min of incubation at 4°C, cells were washed twice, and bound Ab was detected using a FACSCaliber flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

Cell adhesion assays

Immulon 2HB plates (96 wells; Dynex Technologies, Chantilly, VA) were incubated with indicated concentrations of VCAM-1 or the CS-1-containing fragment of fibronectin overnight at 4°C. Afterward, wells were blocked with 2% BSA in PBS for 30 min at room temperature. Cells in modified Tyrode’s buffer were added to wells and allowed to adhere for 40 min at 37°C. Nonadherent cells were washed off with Tyrode’s buffer. Adherent cells were stained with 0.5% crystal violet stain in 20% methanol. The cell-incorporated crystal violet was solubilized with 10% acetic acid and measured in a microplate reader (Molecular Devices, Sunnyvale, CA) set at 560 nm.

Cell migration assay

Cell migration was assayed in a modified Boyden chamber assay system. Transwells (Costar) polycarbonate membranes containing 3.0-µm pores were incubated with VCAM-1 and/or ICAM-1 in 0.1 M NaHCO3, pH 8.0, overnight at 4°C. Membranes were blocked with 2% BSA in PBS for 30 min at room temperature. To the top chamber, 2.0 x 105 cells in RPMI 1640 with 10% FCS were added. Stromal-derived factor-1{alpha} (SDF-1{alpha}; R&D Systems, Minneapolis, MN) at a final concentration of 15 ng/ml in RPMI 1640 with 10% FCS was added to the bottom chamber. Cells were allowed to migrate for 4 h at 37°C. Cells in the bottom chamber were enumerated with a hemocytometer.

In experiments using transiently transfected cells, the migration was corrected for transfection efficiency as follows. Briefly, cells were transfected by electroporation as described with cDNAs encoding proteins of interest plus a vector (pEGFP) encoding green-fluorescent protein (GFP; BD Biosciences Clontech, Palo Alto, CA) at a 3:1 ratio, respectively. Cells were assessed for GFP expression by flow cytometry before and after migration. Percent GFP positive values were used to correct for transfection efficiency.

Western blotting

For analysis of FAK and Pyk-2, Jurkat cells were harvested, resuspended at 5 x 106 cells/ml in RPMI without serum (supplemented with 1 mg/ml BSA), and allowed to stand at rom temperature for 1 h before use. Six-well tissue culture plates were precoated with 5 µg/ml GST-CS-1 in PBS (Ca/Mg free, pH 8.0) for 1 h and then blocked with BSA (1 mg/ml) for at least 30 min. Jurkat cells were plated (1 ml) and incubated (37°C, 6% CO2) for 60 min. After incubation, both nonadherent and adherent cells were flash frozen in liquid nitrogen. Cells were then lysed in modified radioimmunoprecipitation buffer: 50 mM Tris pH (7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 1 mM Na3VO4, and a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). To 200 µg of cell lysate, 2 µg of polyclonal anti-Pyk2 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-FAK (BD PharMingen, San Diego, CA) Abs were added and rotated for at least 2 h at 4°C. Packed protein G-Sepharose (10 µl; LKB; Pharmacia, Peapack, NJ) was then added for 2 h and rotated at 4°C. Beads were washed twice in modified radioimmunoprecipitation buffer. Bound proteins were eluted by boiling in reducing SDS-PAGE sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. Nitrocellulose membranes were then blocked in 5% milk, 20 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20; incubated with primary Ab for 1 h at room temperature; washed; and then incubated with goat anti-mouse HRP-conjugated secondary Ab (Biosource, Camarillo, CA). Bound Ab was detected by chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

For analysis of transfected proteins, Jurkat cells were lysed 48 h after transfection as described above. Whole cell lysates were boiled in reducing SDS-PAGE buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were subsequently probed for either paxillin with a rabbit polyclonal Ab. HA- or Myc-tagged protein was probed with respective mAbs. Bound Ab was detected with HRP-conjugated secondary Abs and ECL chemiluminescence as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The paxillin-binding site of the {alpha}4 tail is required for integrin {alpha}4{beta}1 trans-regulation of {alpha}L{beta}2

Ligation of integrin {alpha}4{beta}1 markedly stimulates {alpha}L{beta}2-mediated migration in T cells (8). To assess the role of paxillin binding in this process, we first examined the effect of disruption of the paxillin-binding site of the {alpha}4 tail. To do this, we derived {alpha}4-deficient Jurkat T cells by chemical mutagenesis and FACS that could be reconstituted with {alpha}4 (Fig. 1). These cells were then transfected with either {alpha}4Wt or the {alpha}4(Y991A) mutant that lacks paxillin-binding function (15, 16) and stable lines isolated by FACS. Both lines express similar levels of {alpha}4, {alpha}5, {beta}1, and {beta}2 subunits (Fig. 1). Furthermore, because the reconstituted cells are derived from the same parent {alpha}4-deficient Jurkat cell, they will share any extraneous mutations introduced during the ethyl methanesulfonate mutagenesis. On an ICAM-1 substrate, the migration of cells reconstituted with {alpha}4Wt was markedly stimulated by the presence of small quantities of VCAM-1 (Fig. 2A). The stimulated migration was largely blocked with an anti-{beta}2 Ab, TS1/18, indicating that the migration was {beta}2 integrin dependent. In addition, the stimulated migration was blocked with an anti-VCAM-1 Ab, P8B1, indicating its VCAM-1 dependence. The {alpha}4(Y991A) mutation abrogated the capacity of {alpha}4 to stimulate {alpha}L{beta}2-dependent cell migration (Fig. 2B). However, the cells bearing the Y991A mutation did not have a global defect in cell migration because they migrated as well as cells bearing {alpha}4Wt on a pure ICAM-1 substrate (Fig. 2C). Consequently, a mutation that disrupts paxillin binding to integrin {alpha}4 interferes with the capacity of {alpha}4{beta}1 to stimulate {alpha}L{beta}2-dependent migration.



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  		FIGURE 1. Integrin expression on reconstituted Jurkat T cells. The generation of {alpha}4 integrin-deficient Jurkat T cells and their reconstitution with {alpha}4Wt or {alpha}4 with an alanine substitution at Tyr991 (Y991A) is described in Materials and Methods. Cells were stained with anti-{alpha}4 (HP2/1), anti-{alpha}5 (SAM1), anti-{beta}1 (K20), or anti-{beta}2 (TS1/18) Abs all at 10 µg/ml. Bound Ab was detected with a FITC-conjugated goat anti-mouse IgG by flow cytometry. Solid histograms depict binding of control mouse IgG. Open histograms show binding of the indicated anti-integrin Ab. Mean fluorescence intensity (MFI) is indicated for each staining. Results are representative of three separate experiments.

 


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  		FIGURE 2. Disruption of the paxillin-binding site of {alpha}4 integrin interferes with {alpha}4{beta}1-stimulated {alpha}L{beta}2-dependent cell migration. {alpha}4-deficient Jurkat T cells were stably reconstituted with {alpha}4Wt or {alpha}4 with an alanine substitution at Tyr991 ({alpha}4(Y991A)) which disrupts paxillin binding. Migration assays were performed as described in Materials and Methods. Transwells were coated with ICAM-Ig (10 µg/ml) and the indicated concentrations of VCAM-Ig. Jurkat cells expressing {alpha}4Wt (A) or {alpha}4(Y991A) (B) were added to the top chamber either untreated or treated with anti-{beta}2 Ab (TS1/18; 20 µg/ml) or anti-VCAM-1 (P8B1; 20 µg/ml). SDF-1{alpha} (15 ng/ml) was added to the bottom chamber, and cells were allowed to migrate for 4 h at 37°C. Cells in the bottom chamber were enumerated, and migration was expressed as a percent change relative to no VCAM-1 addition. Similar results were observed when the ICAM-1 coating concentration was 200 µg/ml. C, Transwell membranes were coated with the indicated concentrations of ICAM-Ig, and migration assays were performed as described above. Cells migrating to the bottom chamber were enumerated with a hemocytometer, and migration was expressed as a percent of input cells. Results are the mean ± SEM of three separate experiments.

 
These reductions in {alpha}4-dependent migration in cells expressing {alpha}4(Y991A) were not due to differences in {alpha}4{beta}1 or {beta}2 integrin expression (Fig. 1). Furthermore, we previously found that the affinity of integrin {alpha}4 regulates its ability to stimulate {beta}2-dependent migration (8). However, the cells expressing either {alpha}4Wt or {alpha}4(Y991A) bound soluble VCAM-1 with similar affinity (Fig. 3A). In addition, cell adhesion to VCAM-1 or the CS-1 region of FN was not altered by the {alpha}4(Y991A) mutation (Fig. 3B). Consequently, the failure {alpha}4(Y991A) to support trans-regulation is not due to a reduction in ligand-binding affinity or cell adhesion. It is therefore ascribable to a defect in the capacity of that receptor to transduce events that follow integrin engagement.



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  		FIGURE 3. Disruption of paxillin binding to {alpha}4 integrin does not affect {alpha}4-dependent soluble ligand binding or static cell adhesion. A, Soluble (s) VCAM-1 binding to Jurkat cells expressing {alpha}4Wt ({blacksquare}, {square}) or {alpha}4(Y991A) (•, {circ}). Cells were incubated with the indicated concentrations of soluble VCAM-1 in buffer containing Ca2+ and Mg2+ ({circ}, {square}) or Mn2+ (•, {blacksquare}) as an exogenous integrin activator. Bound VCAM-1 was detected with a FITC-conjugated goat anti-human IgG by flow cytometry and expressed as mean fluorescence intensity (MFI). B, Static Jurkat cell adhesion to VCAM-1 or CS-1 fragment of fibronectin (CS-1-FN) as ligands for {alpha}4{beta}1. Plates containing 96 wells were coated with the indicated concentration of either VCAM-1 (left) or CS-1-FN (right). Cells were allowed to adhere for 40 min at 37°C. Cell adhesion was quantified by crystal violet staining and expressed as a percent of input cell. Results are representative of three separate experiments.

 
An {alpha}4-binding fragment of paxillin inhibits integrin {alpha}4{beta}1 trans-regulation of {alpha}L{beta}2

The effect of the {alpha}4(Y991A) mutation provides evidence for the role of paxillin in {alpha}4 stimulation of {beta}2-dependent cell migration. However, it is possible that the mutation interfered with {alpha}4 functions other than paxillin binding. Consequently, we sought an alternative means to test the role of paxillin binding in this process. A fragment of paxillin containing residues 176–277 (Pax176–277) binds to {alpha}4 and competes with full-length paxillin for {alpha}4 binding (26). Transfection of a cDNA encoding Pax176–277 profoundly inhibited the {alpha}4-stimulated {beta}2-dependent migration of Jurkat cells (Fig. 4A). In contrast, transfection of a non-{alpha}4-binding fragment of paxillin (Pax1–125) resulted in negligible inhibition. Furthermore, Pax176–277 had much less effect on {beta}2-dependent cell migration on ICAM-1 alone. Thus, its effects are relatively specific for {alpha}4-stimulated migration. Western blot analysis revealed that both paxillin fragments were well expressed (Fig. 4B). In sum, a paxillin fragment that blocks the binding of intact paxillin to {alpha}4 inhibited VCAM-1 stimulation of T cell migration on ICAM-1.



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  		FIGURE 4. Expression of an {alpha}4 binding fragment of paxllin inhibits {alpha}4{beta}1-stimulated {alpha}L{beta}2-dependent Jurkat cell migration. A, Jurkat cells were transiently transfected with cDNAs encoding an {alpha}4-binding region of paxillin (aa 176–277; Pax176–277), a non-{alpha}4-binding fragment of paxillin (aa 1–125; Pax1–125), or vector control DNA along with a GFP-encoding vector as a marker of transfection. Cells were allowed to migrate across Transwell membranes coated with ICAM-1 (200 µg/ml) with or without the addition of VCAM-1 (1 µg/ml) for 4 h at 37°C as described in Fig. 2. Total cells migrating to the bottom chamber were enumerated with a hemocytometer and corrected for GFP expression as measured by flow cytometry. Migration is expressed as a percent inhibition relative to empty vector-transfected cells. Results are mean ± SEM of three separate experiments. B, Immunoblot for paxillin. Cells were transfected as described above, and whole cell lysates were prepared. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting for paxillin. Endogenous full length paxillin is seen at ~62 kDa (KD), and the transfected paxillin fragments are seen between 17 and 30 kDa. Results are representative of three separate experiments.

 
Binding of paxillin to the {alpha}4 cytoplasmic domain stimulates integrin {alpha}4{beta}1-dependent T cell migration on a pure VCAM-1 substrate

The foregoing experiments analyzed the role of paxillin binding in the capacity of {alpha}4 integrins to stimulate {alpha}L{beta}2-dependent cell migration. We also examined the role of paxillin binding in the ability of {alpha}4{beta}1 to directly mediate T cell migration on a pure VCAM-1 substrate. Only one-third as many {alpha}4(Y991A) Jurkat cells migrated across filters coated with 10 µg/ml VCAM-1 as did the cells expressing {alpha}4Wt, a 65% reduction in directed migration (Fig. 5A). Furthermore, transfection of Pax176–277 also suppressed migration on VCAM-1 (Fig. 5B). Thus, both direct {alpha}4 integrin-mediated migration and {alpha}4-stimulated {alpha}L{beta}2-dependent migration of T cells is inhibited by blockade of paxillin binding to the {alpha}4 tail.



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  		FIGURE 5. Paxillin binding promotes {alpha}4{beta}1-mediated migration of Jurkat cells on VCAM-1. A, Transwell membranes were coated with the indicated concentrations of VCAM-1. Jurkat cells expressing {alpha}4Wt ({blacksquare}) or {alpha}4(Y991A) ({diamondsuit}) were added to the top chamber, and SDF-1{alpha} (15 ng/ml) was added to the bottom chamber. Cells migrating to the bottom chamber were enumerated with a hemocytometer and expressed as a percent of input cells. B, Jurkat cells were transiently transfected with cDNAs encoding an {alpha}4-binding region of paxillin (aa 176–277; Pax176–277), a non-{alpha}4-binding fragment of paxillin (amino acids 1–125; Pax1–125), or vector control DNA along with a GFP-encoding vector as a marker of transfection. Cells were allowed to migrate across Transwell membranes coated with VCAM-1 (10 µg/ml) for 4 h at 37°C as described in Fig. 2. Total cells migrating to the bottom chamber were enumerated with a hemocytometer and corrected for GFP expression as measured by flow cytometry. Migration is expressed as a percent inhibition relative to empty vector-transfected cells. Results are the mean ± SEM of three separate experiments.

 
Paxillin binding to {alpha}4 promotes the activation of the tyrosine kinases, Pyk2 and FAK

Pyk2 and FAK are related tyrosine kinases that are tight binding partners of paxillin and are implicated in regulating cell migration (17, 18, 19, 20, 21, 22). Thus, we examined the involvement of these kinases in {alpha}4{beta}1 enhancement of {alpha}L{beta}2-dependent cell migration. Jurkat cells expressed Pyk2 and FAK (Fig. 6). {alpha}4-Mediated adhesion of Jurkat cells expressing {alpha}4(Y991A) to VCAM-1 alone resulted in markedly reduced Pyk2 and FAK tyrosine phosphorylation when compared with cells expressing {alpha}4Wt (Fig. 6). Little Pyk2 and FAK stimulation was observed on exposure to ICAM-1 alone, which may reflect the low constitutive adhesion to ICAM-1. However, addition of trace amounts of VCAM-1 to the ICAM-1 substrate stimulated Pyk2 and FAK tyrosine phosphorylation, which again was attenuated greatly in the {alpha}4(Y991A)-expressing cells. Thus, the integrity of the paxillin-binding site is required for optimal Pyk2 and FAK activation by {alpha}4 integrins.



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  		FIGURE 6. The interaction of paxillin with {alpha}4 integrin enhances Pyk2 and FAK tyrosine phosphorylation in Jurkat T cells. Jurkat cells expressing {alpha}4Wt or {alpha}4(Y991A) (Y/A) were kept in suspension (Susp) or allowed to adhere to plates coated with the VCAM-1 (10 µg/ml), ICAM-1 (10 mg/ml), or ICAM-1 (10 µg/ml) + VCAM-1 (0. 5 µg/ml) for 60 min. Pyk2 and FAK were immunoprecipitated from cell lysates and analyzed by SDS-PAGE. Immunoblots were probed with an anti-phosphotyrosine Ab followed by an anti-Pyk2 or anti-FAK Ab. Bound Ab was detected with HRP-conjugated secondary Abs and chemiluminescence. IP, immunoprecipitation; WB, Western blot. Results shown are representative of three separate experiments.

 
FAK and/or Pyk2 mediate paxillin-dependent integrin {alpha}4{beta}1 trans-regulation of {alpha}L{beta}2

The C-terminal domains of Pyk2 and FAK are essential for correct cellular localization and function of these kinases (27, 28). To determine whether Pyk2 and FAK functions are involved in {alpha}4{beta}1-stimulated {beta}2-dependent migration, Jurkat cells were transfected with C-terminal fragments of Pyk2 and FAK that lack the kinase domain. These constructs, PRNK and FRNK, respectively, act as dominant-interfering mutants of these kinases (29, 30, 31, 32). Both FRNK and PRNK strongly inhibited {alpha}4{beta}1-stimulated {beta}2-dependent cell migration (Fig. 7A). This inhibition required the capacity of the fragments to bind to paxillin, because a variant of FRNK with a mutation that disrupts paxillin binding (33), FRNK-L/S, was much less effective at blocking migration (Fig. 7A) even though it was well expressed (Fig. 7B). Thus, the functions of Pyk2 and/or FAK are required for trans-regulation between integrin {alpha}4{beta}1 and {alpha}L{beta}2.



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  		FIGURE 7. FAK and/or Pyk2 are required for {alpha}4{beta}1- stimulated {alpha}L{beta}2-dependent Jurkat cell migration. A, PRNK and FRNK inhibit {alpha}4{beta}1-stimulated {alpha}L{beta}2-dependnt migration. Jurkat cells were transiently transfected with cDNAs encoding PRNK, FRNK, FRNK with a mutation that disrupts paxillin binding (FRNK L/S), or vector control DNA in combinations with a vector encoding GFP as a marker of transfection. Cells were allowed to migrate across Transwell membranes coated with ICAM-1 (200 µg/ml) with or without the addition of VCAM-1 (1 µg/ml) for 4 h at 37°C as described in Fig. 1. Total cells migrating to the bottom chamber were enumerated with a hemocytometer and corrected for GFP expression as measured by flow cytometry. Migration is expressed as a percent inhibition relative to empty vector-transfected cells. Results are the mean ± SEM of three separate experiments. B, Whole cell lysates were prepared from cells as described above. Lysates were separated by SDS-PAGE and immunoblotted with an anti-HA Ab (12CA5) (left) or anti-Myc Ab (9E10) (right).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Engagement of integrin {alpha}4{beta}1 markedly stimulates leukocyte migration mediated by integrin {alpha}L{beta}2. We now report that this form of integrin trans-regulation in T cells requires the binding of paxillin to the {alpha}4 integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by a mutation that disrupts paxillin binding. Furthermore, cellular expression of a paxillin fragment that blocks {alpha}4 binding to paxillin also inhibited {alpha}4{beta}1 stimulation of {beta}2-dependent migration. The {alpha}4-paxillin association mediates cross-talk by enhancing the activation of the related tyrosine kinases, FAK and Pyk2, based on two lines of evidence. First, {alpha}4{beta}1-mediated adhesion of Jurkat cells bearing {alpha}4(Y991A) led to much less phosphorylation of Pyk2 and FAK relative to cells bearing wild-type {alpha}4. Secondly, C-terminal fragments of Pyk2 and FAK, which lack the kinase domain and function as dominant negative variants, blocked {alpha}4{beta}1 stimulation of {beta}2-dependent migration. Thus, communication between {alpha}4{beta}1 and {alpha}L{beta}2 integrins requires the interaction of paxillin with {alpha}4 and the subsequent accelerated activation of FAK and/or Pyk2. These studies define a specific interaction of an integrin cytoplasmic domain required for integrin trans-regulation.

The {alpha}4 integrin-paxillin interaction is required for trans-regulation between {alpha}4{beta}1 and {alpha}L{beta}2 integrins. This conclusion is based on two distinct experimental approaches. First, a point mutation in the {alpha}4 cytoplasmic domain that disrupts paxillin binding interferes with {alpha}4{beta}1 stimulation of {beta}2-dependent migration. Previously, this mutation selectively blocked the binding of paxillin but not several other proteins to the {alpha}4 tail (15, 16). This selectivity suggests that the effects of the Y991A mutation can be ascribed to the disruption of the interaction with paxillin. Furthermore, an {alpha}4-binding fragment of paxillin blocks paxillin binding to {alpha}4. Transfection of Jurkat cells with a cDNA encoding this fragment inhibited {alpha}4{beta}1 enhancement of {alpha}L{beta}2 cell migration. Thus, based on two independent lines of evidence, the direct binding of paxillin to {alpha}4 is required for {alpha}4{beta}1 enhancement of {beta}2 integrin-dependent cell migration.

The effect of paxillin binding on trans-regulation between {alpha}4{beta}1 and {alpha}L{beta}2 is due to alterations in events that follow the binding of ligands to {alpha}4{beta}1. The capacity of VCAM-1 binding to integrin {alpha}4{beta}1 to stimulate {beta}2 integrin-dependent migration is a function of the affinity state of {alpha}4{beta}1 (8). However, the Y991A mutation that disrupts paxillin binding did not alter the affinity of integrin as evidenced by undiminished binding of soluble VCAM-1. Furthermore, this mutation did not inhibit static cell adhesion to VCAM-1. Thus, paxillin binding to the {alpha}4 tail is required for events subsequent to adhesion that enhance {alpha}L{beta}2-dependent cell migration on ICAM-1.

FAK and Pyk2 are paxillin binding tyrosine kinases required for trans-regulation between {alpha}4{beta}1 and {alpha}L{beta}2. We found that the binding of the {alpha}4 tail to paxillin led to a substantial increase in the phosphorylation of FAK and Pyk2 following {alpha}4{beta}1-dependent cell adhesion. FAK/Pyk2 phosphorylation is indicative of their activation and that of Src kinases (34). Both FAK/Pyk2 and Src family kinase activation promote integrin-dependent cell migration (20, 21, 22, 35, 36). In addition, cellular expression of C-terminal dominant negative variants of FAK and Pyk2 (FRNK and PRNK), which lack the kinase domain, blocked the {alpha}4{beta}1 enhancement of {beta}2-dependent cell migration. The capacity of FRNK to inhibit required the integrity of its paxillin-binding site. FRNK is not likely to perturb the binding of {alpha}4 to paxillin, because FAK and {alpha}4 can form a ternary complex with paxillin (26). Furthermore, FRNK is known to block the localization of FAK to paxillin-rich sites in cells and to disassemble the FAK-paxillin complex (30, 37). Thus, we favor the explanation that FRNK and PRNK block trans-regulation by disrupting the association of Pyk2 and FAK with paxillin. This explanation implies that the {alpha}4-paxillin interaction recruits FAK and Pyk2 to sites of {alpha}4-mediated adhesion and suggests that the regulation of the Pyk2/FAK interaction with paxillin could be another control point in the regulation of {alpha}4 integrin-induced trans-regulation. Phosphorylated FAK and Pyk2 form docking sites for activated Src kinases; the latter kinases are myristoylated and thus preferentially colocalized with urokinase plasminogen activator receptor in lipid microdomains (27, 28, 38, 39). Thus, the capacity of {alpha}4 to bind to a paxillin-FAK complex raises the possibility that this interaction contributes to urokinase plasminogen activator receptor-dependent enhancement of {alpha}L{beta}2-dependent cell adhesion (7). Furthermore, the movement of {alpha}L{beta}2 into lipid rafts has been proposed as one means of regulating its function, and this is thought to occur through disruption of cytoskeletal constraints (40). Consequently, a distal component of {alpha}4 integrin-mediated trans-regulation of {beta}2-dependent migration may be subregional translocation in the plasma membrane. In any case, our data establish that the {alpha}4-paxillin interaction stimulates {beta}2-depedendent migration by promoting the activation of Pyk2 and/or FAK.

Cooperation between integrins {alpha}4{beta}1 and {alpha}L{beta}2 may be involved in specifying preferred sites for the recruitment of leukocytes to sites of inflammation. ICAM-1, a ligand for integrin {alpha}L{beta}2, is widely and constitutively expressed on the vascular endothelium (1). In contrast, VCAM-1 is expressed only at selected sites including sites of inflammation (1). VCAM-1 can engage {alpha}4{beta}1 leading to markedly enhanced {alpha}L{beta}2-dependent cell migration, and the sensitivity of cells to this form of stimulation depends on the affinity of {alpha}4{beta}1 (8). Migration can be stimulated by signaling mechanisms or by {alpha}4{beta}1-mediated lateral movement followed by {alpha}L{beta}2-dependent diapedesis (8, 41). We report here that paxillin binding to the {alpha}4 tail is required for both efficient communication between {alpha}4{beta}1 and {alpha}L{beta}2 and for direct {alpha}4{beta}1-dependent migration. Thus, these studies define a specific interaction of an integrin cytoplasmic domain required for multiple forms of {alpha}4-dependent leukocyte migration.


   Acknowledgments
 
We thank The Scripps Research Institute Flow Cytometry Core Facility for help in the isolation of Jurkat cell variants and The National Cell Culture Center for assistance in the isolation and purification of the recombinant VCAM-Ig and ICAM-Ig fusion proteins. This is publication 14842-CB from The Scripps Research Institute.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AR27214, HL59007, and HL48728 (to M.H.G.), grants from the Arthritis Foundation (to D.M.R.) and the Juvenile Diabetes Research Foundation (to D.M.R.), and National Institutes of Health Grant P30 AR47360 (to D.M.R.). Back

2 D.M.R. and S.L. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. David M. Rose, VA Medical Center, 3350 La Jolla Village Drive, MC111K, San Diego, CA 92161. E-mail address: drose{at}vapop.ucsd.edu Back

4 Abbreviations used in this paper: VLA-4, very late Ag-4; SDF-1{alpha}, stromal-derived factor-1{alpha}, FAK, focal adhesion kinase; FRNK, FAK-related nonkinase; FRNK-L/S, FRNK-leucine1034serine; Pyk-2, proline-rich tyrosine kinase-2; PRNK, Pyk-2-related nonkinase; {alpha}4Wt, wild-type {alpha}4; GFP, green-fluorescent protein; HA, hemagglutinin. Back

Received for publication August 26, 2002. Accepted for publication April 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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