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Cutting Edge: A Novel Function for the SLAP-130/FYB Adapter Protein in ß₁ Integrin Signaling and T Lymphocyte Migration¹

Anne J. Hunter^*, Nadine Ottoson^*, Nancy Boerth, Gary A. Koretzky and Yoji Shimizu^2,*

^* Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis, MN 55455; and Department of Pathology and Lab Medicine, Leonard and Madlyn Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that ₄ß₁ integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the ₄ß₁ integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59^fyn. Activation of normal T cells, but not H9 T cells, via ₄ß₁ leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in ß₁ integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Orchestration of T lymphocyte migration involves a multistep process requiring signals provided by integrin receptors (1, 2). Although ß₁ integrins transduce intracellular signals upon ligand engagement that can enhance organization of the cytoskeleton, T cell activation, and induce transcriptional events in the nucleus (3, 4, 5), little is known about the role of ß₁ integrin-mediated signaling in lymphocyte migration. Since there is clear convergence in signaling between integrins and growth factor receptors (5), we have explored the role in ß₁ integrin signaling of proteins that become tyrosine phosphorylated following TCR stimulation (6, 7). One such substrate is the SH2 domain-containing 76-kDa protein (SLP-76)³-associated phosphoprotein of 130 kDa (SLAP-130) (8) or p59^fyn-binding protein (Fyb) (9), which can associate with the adapter protein SLP-76 (8), the src family tyrosine kinase p59^fyn (9), and Src kinase-associated phosphoprotein of 55 kDa (SKAP 55) (10, 11). Although SLP-76 clearly plays a vital role in T cell development (12, 13), TCR-induced transcriptional activation of the IL-2 gene (14, 15), and TCR-mediated regulation of the cytoskeleton (16), the role of SLAP-130/FYB in TCR signaling is much less clear (8, 9). In this report, we describe a novel role for SLAP-130/FYB in ß₁ integrin signaling and ß₁ integrin-dependent T cell migration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cells and culture reagents

H9 T cells (provided by Dr. P. Kavathas, Yale University, New Haven, CT) were maintained in RPMI 1640 medium containing 10% FCS (Atlanta Biologicals, Atlanta, GA), L-glutamine, and 1% penicillin/streptomycin. PHA-activated human T cell blasts were prepared as previously described (17).

Abs and other reagents

The anti-SLAP-130 and anti-SLP-76 Abs have been described previously (8). The anti-phosphotyrosine (ptyr) mAb 4G10 was purchased from Upstate Biotechnologies (Lake Placid, NY). The anti-₄ integrin mAb NIH49d-1 was provided by Dr. S. Shaw (National Institutes of Health, Bethesda, MD). The anti-CD3 mAb OKT3, the anti-ß₁ integrin mAb TS2/16, and the anti-MHC class I mAb W6/32 were purchased from the American Type Culture Collection (Manassas, VA). The inhibitory ß₁ integrin-specific mAb AIIB2 was purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). The anti-CXCR4 mAb was purchased from R&D Systems (Minneapolis, MN). The GST fusion protein expressing the SH2 domain of fyn was provided by Drs. R. Herrera and S. Hubbell (Warner-Lambert/Parke-Davis, Ann Arbor, MI). PMA was purchased from Sigma (St. Louis, MO), dissolved in DMSO (100 µg/ml) and stored at -70°C. Human fibronectin (FN) was provided by Dr. J. McCarthy (University of Minnesota, Minneapolis, MN).

Preparation of cell lysates

Cell lysates were prepared as previously described (18). Briefly, H9 T cells or human T cell blasts were harvested and incubated with 2 µg/10⁶ cells of the indicated primary mAb for 30 min at 4°C, washed, and then incubated with 0.5 µg/10⁶ cells of goat anti-mouse IgG (Organon Teknika, Malvern, PA) for 15 min at 4°C. The cells were then incubated in a 37°C water bath from 0 to 30 min and lysed by adding an equivalent volume of 2x lysis buffer (18). Unstimulated cells were incubated with goat anti-mouse IgG and lysed as described above. To assess FN-induced phosphorylation events, H9 T cells were plated on six-well tissue culture plates precoated with either poly-L-lysine (PLL) or human FN (30 µg/ml) for 5 min at 37°C in the presence of 250 µM Mn²⁺, which induces the high affinity form of ₄ß₁ integrin. Cells were harvested and lysed as above.

Immunoprecipitation

Immunoprecipitations were performed as previously described (18) using goat anti-mouse IgG (H+L) Sepharose (Zymed, San Francisco CA) or GammaBind Plus Sepharose (Pharmacia Biotech, Uppsala, Sweden) (50 µl per precipitation) precoated with 5 µl anti-SLAP-130 or anti-SLP-76 Ab. Cell lysates (10 x 10⁶ H9 cells or 20 x 10⁶ human T cells) were then incubated with the Ab-coated beads, washed in lysis buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) for analysis by Western blotting.

GST-SH2 fusion protein precipitation

Glutathione-Sepharose-conjugated GST-SH2 fusion proteins (10 µg) were incubated with cell lysate (10 x 10⁶ cell equivalents) overnight at 4°C, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes for analysis by Western blotting as previously described (18).

Western blotting

Western blotting was performed as previously described (18). Blots were incubated with primary Ab (1:500 dilution of anti-SLAP-130 or anti-SLP-76 in 2.5% BSA or 0.5 µg/ml anti-Ptyr mAb 4G10 in 5% milk) for 2 h at room temperature, washed, and then incubated with HRP-conjugated goat anti-mouse IgG (Caltag, South San Francisco, CA) or rabbit anti-sheep IgG (H+L) (Bio-Rad, Hercules,CA) for 1 h at room temperature. The membranes were washed as previously described (18) and developed using enhanced chemiluminescence (Pierce, Rockford, IL). For repeated immunoblotting, membranes were stripped by incubating the membrane in 62.5 mM Tris-HCl (pH 6.7), 0.1 M 2-ME, and 2% SDS for 45 min in a 50°C water bath. The membranes were rinsed with PBS containing 0.1% Tween 20 and blocked with PBS containing 5% milk before reprobing using the blotting procedure described above.

Construction of green fluorescent protein (GFP)-SLAP-130/FYB plasmid expression vector

A 2.2-kb FLAG-tagged SLAP-130/FYB cDNA fragment from the pEF/SLAP-130/FYB vector (8) was subcloned in frame into the pEGFP-C1 plasmid (Clontech, Palo Alto, CA) to produce the pEGFP-SLAP-130/FYB expression vector. This vector encodes for a GFP-SLAP-130/FYB fusion protein where SLAP-130/FYB has been fused to the carboxyl-terminal end of GFP.

Transient transfections

T cell blasts (harvested on day 3 or 4) were transfected with either pEGFP-C1 or pEGFP-SLAP-130/FYB as previously described (17). Cells were harvested after 16–18 h and used either in a migration assay or to detect expression of GFP fusion by Western blot analysis.

Migration assay

Migration assays were performed as previously described (19) using Transwell chambers with 3-µm polycarbonate filters (Costar, Cambridge, MA; cat. no. 3414) coated with 20 µg/ml FN. Human SDF-1 (Peprotech, Rocky Hill, NJ) was diluted to a concentration of 100 ng/ml in migration assay media (RPMI supplemented with 1% FCS, pH 7.0) and added to the lower chamber of the transwells. The FN-coated transwell inserts were placed on top, and 2 x 10⁶ T cells in 1 ml migration assay media was added to the upper chamber. After 3 h of incubation at 37°C, cells that had migrated through the filter were collected, pelleted, and resuspended in 200 µl of FACS buffer (HBSS supplemented with 10% bovine calf serum and 0.2% sodium azide). PKH26 reference microbeads (50 µl) (Sigma) and 25 µl of propidium iodide (Sigma) were added to each tube and samples were analyzed by flow cytometry (19). An aliquot of each transfected population was also analyzed by single-color flow cytometry using standard procedures to assess expression of CXCR4 (3). Quantitation of cell migration was determined as previously described (19) by calculating the total number of T cells in each sample. Initial numbers of T cells added to each well at the start of the migration assay were calculated by the same procedure using premigration cell samples. Postacquisition gating based on GFP (FL1) expression was used to determine the mean percentage of migration of GFP-negative and GFP-positive T cells from the samples in the absence or presence of SDF-1.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
To determine whether stimulation of ₄ß₁ integrins leads to tyrosine phosphorylation of SLAP-130/FYB, H9 T cells were stimulated with the ₄ integrin-specific mAb NIH49d-1 for 0 to 20 min, and anti-Ptyr immunoblotting was performed on anti-SLAP-130/FYB immunoprecipitates. Fig. 1A shows that ₄ integrin stimulation leads to tyrosine phosphorylation of SLAP-130/FYB that was detectable within 30 s of stimulation, peaked between 2 and 5 min, and returned to basal levels by 10 min. Reprobing of the blot with the anti-SLAP-130 Ab demonstrated that comparable amounts of SLAP-130/FYB were present in each sample, except for a slight reduction in SLAP-130/FYB at 10 and 20 min following ₄ integrin stimulation (Fig. 1A). Similar results were observed following stimulation with the ß₁ integrin-specific mAb TS2/16 (Fig. 2). In contrast, stimulation of H9 T cells with the anti-MHC class I mAb W6/32 does not lead to tyrosine phosphorylation of SLAP-130/FYB at any of the time points examined, even though MHC class I is expressed at a level comparable to ₄ß₁ on these cells (Fig. 1 and data not shown). Adhesion of H9 T cells to human FN, which is mediated by the ₄ß₁ integrin, also results in enhanced phosphorylation of SLAP-130/FYB when compared with nonspecific adhesion to PLL (Fig. 1B). Thus, SLAP-130/FYB is one of several substrates that can be phosphorylated by both the TCR and ß₁ integrins (20, 21).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. 4ß1 integrin stimulation of H9 T cells results in enhanced tyrosine phosphorylation of SLAP-130/FYB and enhanced association of SLAP-130/FYB with the SH2 domain of fyn. A, H9 T cells were stimulated by cross-linking with the 4 integrin-specific mAb NIH49d-1 and goat anti-mouse IgG as described in Materials and Methods for the indicated periods of time at 37°C. Control stimulation with the anti-MHC class I mAb W6/32 and goat anti-mouse IgG was conducted for 2 min at 37°C. Cell lysates were immunoprecipitated with anti-SLAP-130, separated on a 7.5% SDS-polyacrylamide gel, and immunoblotted with the anti-Ptyr mAb 4G10 (top panel). The membrane was subsequently stripped and reprobed with anti-SLAP-130 (bottom panel). B, H9 T cells were incubated on 6 well plates precoated with PLL or human FN for 5 min in the presence of 250 µM Mn2+. Cell lysates were immunoblotted as described in Panel A. C, H9 T cells were left unstimulated (U) or were stimulated (4) by cross-linking with the 4 integrin-specific mAb NIH49d-1 and goat anti-mouse IgG as in A for 5 min at 37°C. Cell lysates were precipitated with 10 µg of the indicated glutathione-Sepharose-conjugated GST-SH2 fusion proteins or anti-SLAP-130, separated on a 7.5% SDS-polyacrylamide gel, and immunoblotted as in A.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. 4ß1 integrin stimulation of normal human T cell blasts, but not H9 T cells, results in the tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. H9 T cells (A) or human T cell blasts (B) were left unstimulated (U) or were stimulated by cross-linking with either the 4 integrin-specific mAb NIH49d-1, the ß1 integrin-specific mAb TS2/16, or the CD3-specific mAb OKT3 and goat anti-mouse IgG as described in Materials and Methods for 5 or 2 min, respectively, at 37°C. Cell lysates were precipitated with 5 µl of anti-SLAP-130 or anti-SLP-76 Abs, separated on a 7.5% SDS-polyacrylamide gel, and immunoblotted with the anti-Ptyr mAb 4G10 (top panel). The membrane was subsequently stripped and reprobed with anti-SLAP-130 (middle panel) and anti-SLP-76 (bottom panel).

Since TCR stimulation leads to the association of tyrosine phosphorylated SLAP-130/FYB with SLP-76 (8, 9), we examined whether ₄ß₁ integrin stimulation also leads to the association of SLAP-130/FYB with SLP-76. Western blotting analysis of SLP-76 immunoprecipitates showed that CD3 stimulation, but not ₄ß₁ integrin stimulation, of H9 T cells leads to tyrosine phosphorylation of SLP-76 (Fig. 2A). However, a tyrosine phosphorylated substrate that comigrates with SLAP-130/FYB is coprecipitated with SLP-76 in ₄ß₁ integrin- and CD3-stimulated H9 T cells (Fig. 2A). Reprobing with an anti-SLAP-130/FYB Ab demonstrated the presence of SLAP-130/FYB in anti-SLP-76 immunoprecipitates in unstimulated and ₄ß₁ integrin-stimulated H9 T cells. However, the amount of SLAP-130/FYB that coprecipitates with SLP-76 does not increase significantly upon ₄ß₁ integrin stimulation, even though ₄ß₁ integrin stimulation leads to enhanced tyrosine phosphorylation of SLAP-130/FYB. In contrast, CD3 stimulation leads to increased SLAP-130/FYB in SLP-76 immunoprecipitates. This suggests key differences in the phosphorylation of SLAP-130/FYB between ß₁ integrin and TCR stimulation. Anti-Ptyr immunoblotting of anti-SLAP-130/FYB immunoprecipitates revealed that ₄ß₁ integrin stimulation or CD3 stimulation of H9 T cells leads to tyrosine phosphorylation of SLAP-130/FYB, although we consistently observe stronger tyrosine phosphorylation of SLAP-130/FYB upon ₄ß₁ integrin stimulation when compared with CD3 stimulation (Fig. 2A). Since we have been unable to detect SLP-76 in anti-SLAP-130/FYB immunoprecipitates from unstimulated, ₄ß₁ integrin- or CD3-stimulated H9 T cells (Fig. 3A), it is possible that there are other SLP-76-associated proteins that comigrate with SLAP-130/FYB and become tyrosine phosphorylated upon ₄ß₁ integrin stimulation.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Enhancement of T cell migration by SLAP-130/FYB expression in T cell blasts. A, Basal and SDF-1-enhanced migration of human T cell blasts was assessed in the presence of the control anti-MHC class I mAb W6/32 (open bars) or the inhibitory ß1 integrin-specific mAb AIIB2 (hatched bars). B, Whole cell lysates from human T cell blasts transiently transfected with vectors expressing GFP or GFP-SLAP-130/FYB fusion protein were separated on a 7.5% SDS-polyacrylamide gel and immunoblotted with anti-SLAP-130. C, Human T cell blasts transfected (gray histogram) as in (B) were analyzed for GFP (left panel) or GFP-SLAP-130/FYB (right panel) expression by FACS. Untransfected controls (open histogram) were used to determine the GFP- and GFP+ populations. D, Using the gates shown in C, migration of human T cell blasts lacking GFP expression (open bars) or expressing GFP or GFP-SLAP-130/FYB (hatched bars) was assessed as described in Materials and Methods in the absence (top half) or presence of 100 ng/ml SDF-1 (lower half).

Since ß₁ integrin-mediated signal transduction has been implicated in regulating cell migration (22, 23), we investigated a possible role for SLAP-130/FYB in the migration of T cells in response to a chemotactic gradient. We utilized human T cell blasts, which express the CXCR4 chemokine receptor (data not shown). In preliminary experiments, we demonstrated that human T cell blasts migrate in a ß₁ integrin-dependent manner toward the CXCR4 ligand SDF-1 in in vitro migration assays using transwells coated with the ß₁ integrin ligand FN (Fig. 3A). To test the role of SLAP-130/FYB in SDF-1-induced migration, human T cell blasts were transiently transfected with pEGFP-SLAP-130/FYB, which encodes for a GFP-SLAP-130/FYB fusion protein. GFP-SLAP-130/FYB is readily detectable with an anti-SLAP-130 Ab in whole cell lysates from transfected T cell blasts as a 160-kDa protein that can be distinguished from endogenous SLAP-130/FYB, which is detected in GFP-SLAP-130/FYB⁺ transfectants and control cells transiently transfected with the control pEGFP plasmid vector (Fig. 3B). Flow cytometric analysis also revealed expression of GFP and GFP-SLAP-130/FYB in transiently transfected human T cell blasts (Fig. 3C). The level of transfection efficiency was typically 15–20% with human T cell blasts prepared from several different donors. Expression of either GFP or GFP-SLAP-130/FYB did not change the level of expression of either ₄ß₁ integrin or CXCR4 (data not shown).

Human T cell blasts transiently transfected with either pEGFP or pEGFP-SLAP-130/FYB were tested for their ability to migrate through FN-coated transwells in response to SDF-1. Flow cytometry was used to analyze the total number of migrated cells in the bottom chamber, as well as the level of GFP expression in the migrated population. Using the GFP gates shown in Fig. 3C for the original population of transfected T cells that were added to the transwell chambers, the migration of the GFP-negative population was calculated and compared with the migration of the smaller subpopulation of GFP-positive T cells (19). Although there is minimal T cell migration in the absence of SDF-1, migration is enhanced when SDF-1 is added to the bottom of the migration chamber (Fig. 3D). Although T cell blasts expressing GFP alone exhibit levels of migration comparable to their GFP-negative counterparts, there is a 2- to 3-fold increase in the migration of T cell blasts expressing GFP-SLAP-130/FYB. T cells expressing GFP-SLAP-130/FYB also exhibit some increase in migration in the absence of SDF-1 when compared with GFP-negative cells in the same sample. This enhancing effect of SLAP-130/FYB on SDF-1-mediated T cell migration was observed in a minimum of three independent experiments using T cell blasts isolated from different donors. Similar enhancing effects on migration were observed with cotransfection of pEGFP with pEF/SLAP-130/FYB, which encodes for a FLAG-tagged version of SLAP-130/FYB (data not shown).

The enhancement of human peripheral T cell migration upon overexpression of SLAP-130/FYB is similar in some respects to the effects of overexpression of the adapter protein p130^Cas (Cas) on the migration of Chinese hamster ovary (CHO) cells and FG-M tumor cells. In these systems, crk-associated substrate (Cas) has been proposed to enhance cell migration via interactions with focal adhesion kinase (FAK) and the adapter protein crk (23, 24). Our results suggest that SLAP-130/FYB may be a hemopoietic-specific substrate of integrin-mediated signaling that serves to regulate lymphocyte migration. The role of FAK and crk in SLAP-130/FYB-dependent enhancement of T cell migration remains unclear. Unlike Cas, we do not observe ß₁ integrin-dependent association of tyrosine phosphorylated SLAP-130/FYB with crk (data not shown). Although others have reported integrin-mediated tyrosine phosphorylation of FAK in human T cells (25, 26), we have not observed ₄ß₁ integrin-mediated tyrosine phosphorylation of FAK in our T cell systems (18). Another tyrosine kinase may play a role in regulating SLAP-130/FYB-dependent effects on T cell migration. One interesting candidate kinase is fyn, since SLAP-130/FYB can interact with fyn, fyn kinase activity is enhanced upon ₄ß₁ integrin stimulation (18), and fyn can phosphorylate SLAP-130/FYB (27).

In summary, we have identified SLAP-130/FYB as a novel hemopoietic-specific substrate for ₄ß₁ integrin-mediated tyrosine kinase activity. The SLAP-130/FYB binding partner SLP-76 also becomes tyrosine phosphorylated upon ₄ß₁ integrin ligation, although SLP-76 tyrosine phosphorylation varies between normal T cells and a cultured T cell line. The ability of SLAP-130/FYB to enhance T cell migration suggests a novel function for this adapter protein in the regulation of lymphocyte motility.

	Acknowledgments

 
We thank Dr. J. McCarthy for assistance with the migration assays and Dr. M. A. Adelsman for critical comments on the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI38474 and AI31126 (to Y.S.), and GM53256 (to G.K.). A.H. was supported by a predoctoral trainee award from National Institutes of Health Grant AI07313 and by the Program in Signal Transduction and Gene Expression (STAGE) at the University of Minnesota. Y.S. is the Harry Kay Chair of Cancer Research at the University of Minnesota.

² Address correspondence and reprint requests to Dr. Yoji Shimizu, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Box 334 Mayo/6-266 BSBE, 312 Church Street SE, Minneapolis, MN 55455. E-mail address:

³ Abbreviations used in this paper: SLP-76, SH2 domain-containing protein of 76 kDa; SLAP-130, SLP-76-associated phosphoprotein of 130 kDa; Fyn, p59^fyn; FYB, Fyn-binding protein; Cas, crk-associated substrate; FAK, focal adhesion kinase; Ptyr, phosphotyrosine; PLL, poly-L-lysine; FN, fibronectin; SDF, stromal cell-derived factor; EGFP, enhanced green fluorescent protein.

Received for publication September 14, 1999. Accepted for publication November 23, 1999.

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