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Originally published online as doi:10.1189/jlb.0208084 on July 23, 2008

Published online before print July 23, 2008
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(Journal of Leukocyte Biology. 2008;84:1192-1201.)
© 2008 by Society for Leukocyte Biology

Critical role of Lck in L-selectin signaling induced by sulfatides engagement

Ting Xu, Liang Chen, Xin Shang, Lingling Cui, Jixian Luo, Cuixia Chen, Xueqing Ba and Xianlu Zeng1

Institute of Genetics and Cytology, Northeast Normal University, Changchun, P. R. China

1 Correspondence: Institute of Genetics and Cytology, Northeast Normal University, Renmin Street 5268, Changchun, P.R. China. E-mail: zengx779{at}nenu.edu.cn


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ABSTRACT
 
Recruitment of leukocytes onto inflamed tissues is an important physiological event, in which L-selectin plays an essential role in initial leukocyte capture and at the same time, triggers cell signaling. Lck is a member of the Src family of protein tyrosine kinases and is critical for T cell activation triggered by receptor ligation. Here, we demonstrated that Lck was associated directly with and phosphorylated the L-selectin cytoplasmic tail upon L-selectin engagement with sulfatides. Through the direct interaction with ZAP-70 and c-Abl via its Src homology 2 (SH2) and SH3 domains, Lck organized a signaling complex at the cytoplasmic tail of L-selectin. In the cells with Lck knockdown by small interfering RNA treatment, L-selectin signaling was suppressed dramatically, as indicated by reduced phosphorylation of c-Abl and ZAP-70. Re-expression of wild-type or constitutively active but not kinase-dead murine Lck rescued the phosphorylation completely, but the SH2 domain mutant or the SH3/SH2 double mutant of murine Lck had no effect. These results suggest that Lck plays a critical role in L-selectin signaling upon sulfatides stimulation.

Key Words: c-Abl kinase • ZAP-70 kinase • siRNA


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INTRODUCTION
 
Leukocyte recruitment from the bloodstream to a site of inflammation entails a cascade of cellular adhesive events, including tethering, rolling, adhesion, and transmigration. This process is controlled strictly by a series of adhesive molecules expressed on leukocytes and endothelial cells [1 2 3 ]. The selectin family is responsible for the initial tethering and rolling of leukocytes, thereby bringing these cells into proximity with activated endothelium [4 , 5 ]. Extracellular stimuli, together with signals generated by cell–cell interaction, activate integrins, leading to the adhesion and transmigration of leukocytes into tissues [6 ].

Although three members of selectin family can mediate rolling behavior, L-selectin appears to be the major receptor for the early kinetics of leukocyte-endothelial cell interactions [7 8 9 10 11 ]. Besides, a previous study suggested that L-selectin, in particular, contributed in leukocyte migration through its signaling properties [12 ]. It has been reported that in human neutrophils, L-selectin engagement with antibody cross-linking or ligation with ligands could trigger calcium flux, TNF-{alpha}, and IL-8 expression increase [13 ], superoxide generation [14 ], and the activation of NFAT and NF-{kappa}B transcription factors [15 , 16 ]. L-selectin triggering in lymphocytes also results in tyrosine phosphorylation and the activation of MAPK, ERKs [17 ], and Ras protein [18 ]. Through binding with actin-binding protein through its cytoplasmic tail, L-selectin is involved in plasma membrane ruffling and cytoskeleton reorganization [19 , 20 ]. Furthermore, L-selectin clustering induced by mAb cross-linking increased the surface expression of the β2 integrin, which spatially colocalized with L-selectin in the plasma membrane [21 ].

Lck is a member of the Src family of nonreceptor protein tyrosine kinases, which is expressed primarily in T lymphocytes [22 ]. Similar to other members, Lck contains an N-terminal membrane-targeting domain, a unique region, single Src homology 3 (SH3) and SH2 domains, and a tyrosine kinase domain followed by a C-terminal-negative regulatory domain. Lck adopts basal, repressed conformation via the intramolecular SH2 domain binding to the negative regulatory phosphotyrosine residue near the C terminus and the SH3 domain binding to the SH2 kinase linker region, which maintains Lck in an inactive, closed state. On cell activation, these intramolecular interactions cease resulting in the release of the kinase domain, promoting SH2 and SH3 domain-mediated protein–protein interactions that are essential for signal transduction [23 , 24 ].

Lck kinase plays a critical role in transducing signals from the extracellular environment into the cell interior in many different cell types [25 ]. Overexpression of Lck renders T cells hypersensitive to antigen stimulation [26 ], and an Lck-deficient T cell line, J.CaM1, exhibits dramatically reduced protein tyrosine phosphorylation following TCR cross-linking [27 ]. Following TCR engagement, Lck phosphorylates ITAMs in CD3 and TCR{zeta} and enables ZAP-70 to bind [28 ]. ZAP-70 activation induced by Lck kinase promotes the recruitment of downstream signaling molecules. This recruitment initiates a signaling cascade which is required for proliferation and differentiation of developing thymocytes and mature T cells [29 ]. In primary T cells, Lck controls the threshold of cell activation and contributes to the ERK response [30 ]. Furthermore, genetic experiments have shown that mice deficient in Lck or expressing a dominant-negative mutant form of Lck exhibit a severe defect in T cell maturation [31 , 32 ].

L-selectin cross-linking with mAb induces the phosphorylation of Lck, which is responsible for the activation of Ras signaling pathways [18 ]. Our previous work indicated that c-Abl is involved in L-selectin signaling and formed a complex with L-selectin and ZAP-70 [33 ]. Here, we demonstrate that Lck interacts directly with the L-selectin cytoplasmic tail and is activated prior to c-Abl and ZAP-70 upon L-selectin engagement with sulfatides. To further assess the impact of Lck on L-selectin signaling, we use small interfering (si)RNA to specifically and acutely knock down Lck. In cells with Lck knockdown (Lck-KD cells), L-selectin signaling is severely attenuated, and the re-expression of Lck with its kinase activity and intact SH2 domain can rescue this attenuation. These results suggest that Lck is critical for L-selectin signaling induced by sulfatide stimulation.


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MATERIALS AND METHODS
 
Cells, reagents, and plasmids
The human leukemic Jurkat T cells were grown in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% FBS, 10 mM Hepes, 2 mM L-glutamine, 100 U penicillin, and 100 µg/ml streptomycin. In some experiments, antibiotics were removed to improve electroporation. Human embryonic kidney (HEK) 293 cells were maintained in IMDM (Gibco) supplemented with 10% FBS, 100 U penicillin, and 100 µg/ml streptomycin. The primary T lymphocytes and HUVECs were isolated as described previously [34 , 35 ].

Antibody for HLA (W6/32) was obtained from eBioscience (San Diego, CA, USA). Antibodies for Lck (2102), L-selectin (DREG56), ZAP-70 (1E7.2), and c-Abl (K12 and 8E9) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BD Pharmingen (San Diego, CA, USA). Antibody for ZAP-70 Tyr319 (65E4) was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies for phosphotyrosine (PY20), GST (G7781), actin (AC-40), FITC-conjugated phalloidin (P5282), and sulfatides (86125) were obtained from Sigma-Aldrich (St. Louis, MO, USA). STI571 (a special inhibitor to nonreceptor tyrosine kinase c-Abl) was a gift of Novartis Pharma Schweiz AG (Basel, Switzerland). PP2 (the Src family kinase-specific inhibitor) and piceatannol (the Syk/ZAP-70 kinase-specific inhibitor) were obtained from Calbiochem-Novabiochem (San Diego, CA, USA) and Sigma-Aldrich. ECL Plus Western blotting detection reagents (RPN2132) and glutathione (GSH)-sepharose 4BTM (17-0756-01) were purchased from Amersham Biosciences (Piscataway, NJ, USA).

The sequence encoding the cytoplasmic domain of L-selectin was amplified by PCR using the full-length L-selectin cDNA in pcDNA3 as a template. BamHI and EcoRI restriction sites were introduced by the PCR primers, and the fragment was subcloned in pGEX vector. Inactivated mutations of putative phosphorylation sites in the cytoplasmic domain of L-selectin were introduced by using altered PCR primers. The fragments obtained were subcloned into pGEX vectors as described for the wild-type sequence. The plasmids of Lck wild-type and its mutants Y505F, R273A, W97A, R154K, and R154K/W97A were kind gifts from Dr. Hamid Band (Northwestern University, Evanston, IL, USA). The GST-Lck (SH2) and GST-Lck (SH3) were kind gifts from Dr. Ottmar Janssen (Paul-Ehrlich Institute, Langen, Germany ). The GST-c-Abl-NSH and GST-c-Abl-SH2 plasmids were kindly provided by Dr. Nicolas Foray (European Synchrotron Research Facility, France). The GST-CrkII-C-terminal domain (CTD) plasmid was kindly provided by Dr. Giorgio Scita (European Institute of Oncology, Italy). The expression vector expressing ZAP-70 wild-type was kindly provided by Dr. Oreste Acuto (Institute Pasteur, France), and we subcloned the ZAP-70 fragment in the pcDNA3.1 expression vector, which when encoding c-Abl wild-type, was the gift of Dr. Hidesaburo Hanafusa (Rockefeller University, Japan).

siRNA duplexes
The 21-nucleotide siRNA duplexes targeting human Lck mRNA (Lck232; 5'-cugcaagacaaccugguuauc-3'; 5'-uaaccagguugucuugcagug-3') and a triple G/C-mutated control (Lck232M3; 5'-gugcaacacaacgugguuauc-3'; 5'-uaaccacguuguguugcacug-3') have been described previously [36 ] and were synthesized in Gene Pharma Co. Ltd. (Shanghai, China). The oligos were named according to the position of the 5' nucleotide of the sense strand relative to the reference sequences of Lck mRNA (NM_005356).

Cell transfection
Jurkat T cells (20x106) were washed and resuspended in 400 µl OptiMEM (Invitrogen Life Technologies, Carlsbad, CA, USA), mixed with siRNA and/or cDNA and electroporated at 250 V/950 µF in 4 mm cuvettes. Based on previous studies for Lck-KD [36 ], 100 nM siRNA was used, and the cells were incubated for 48 h in complete medium before harvesting. Transfection of HEK293 cells was performed by the calcium-phosphate method, and cells were incubated for 30 h in complete medium before harvesting.

Cell stimulation, lysis, and immunoprecipitations (IPs)
Before stimulations, the Jurkat T cells were preincubated at 37ºC for 5 min. Cells (5x107 cells/ml in RPMI) were stimulated with L-selectin ligand, sulfatides (1 mg/ml), at 37ºC for the indicated periods of time. Cells were lysed in ice-cold lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM each NaF, Na3VO4, and β-glycerolphosphate, and 20 µg/ml aprotinin/leupeptin]. After 30 min incubation on ice and vortexing, the lysates were centrifuged at 13,000 g for 30 min before subjection to SDS-PAGE/immunoblotting or IP. For IPs, the indicated antibodies were added, and incubations continued at 4ºC overnight. Then 20 µl protein A/G sepharose beads (50% slurry) were added to the antibody/lysated mixture for another 3 h. Thereafter, the immune complexes were washed three times in lysis buffer and subjected to SDS-PAGE/immunoblotting. Chemiluminescent detection was performed by using ECL plus Western blotting reagents.

In vitro kinase assay
Jurkat T cells were washed twice in sterile Hepes saline (132 mM NaCl, 20 mM Hepes, 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4) before activation. Cells were stimulated and lysed as mentioned above. Lysates were precipitated with anti-Lck antibody and protein A sepharose beads at 4ºC for 3 h. The beads were subsequently washed at least four times with lysis buffer and followed by three washes with the kinase buffer [25 mM Tris (pH 7.5), 2 mM DTT, 5 mM β-glycerolphosphate, 1 mM Na3VO4, 10 mM MgCl2]. Kinase assays were performed by resuspending the beads in the kinase buffer, adding of 3 µg GST-CytoL and 5 µM cold ATP, and incubating at 30ºC for 30 min. These assays were terminated by adding 20 µl 2x SDS sample buffer. The samples were subjected to SDS-PAGE/immunoblotting.

Pull-down assay
Production of GST and GST-fusion proteins was induced in Escherichia coli strain BL-21 (DE3), transformed with corresponding plasmids by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside at 37ºC for 3 h. Cells were harvested in lysis buffer (20 mM Hepes, pH 7.5, 120 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM PMSF, 10 µg/ml each aprotinin and leupeptin) and homogenized by sonication. Lysates were cleared by centrifugation at 13,000 g at 4ºC for 30 min before being applied to a GSH-sepharose column. After extensive washing with the lysis buffer, the isolated proteins were stored at 4ºC for experiments (not more than 1 week).

Jurkat T cells were stimulated and lysed as mentioned above. Cell lysates were incubated with 10 µl GSH-Sepharose coated with GST or GST-fusion proteins. After 2 h, beads were collected by centrifugation and washed four times with lysis buffer. The bound proteins were eluted by boiling in SDS sample buffer and analyzed by SDS-PAGE.

Far Western assay
Far Western assay was performed as described previously [37 ] with some modifications. Briefly, immunoprecipitates from Jurkat T cells were separated under reducing conditions by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Beford, MA, USA). After nonspecific binding had been blocked with PBS containing 5% nonfat milk, the blots were incubated with blocking buffer containing 10 µg/ml GST or the indicated GST-fusion proteins at 4ºC overnight. Bound proteins were detected by ECL detection.

Immunofluorescence microscopy
Jurkat T cells were stimulated with sulfatides or not after pretreated with PP2, and then the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. The cells were permeabilized with 0.2% Triton X-100 in PBS (containing 5 mM EDTA and 2% FBS) for another 5 min. After washing with PBS, cells were stained with 3.3 x 10–7 M FITC-conjugated phalloidin at room temperature for 20 min. All of the stained cells were observed under the fluorescence microscope.

Flow chamber assay
Rolling of Jurkat T cells to HUVECs was measured under flow conditions by using a parallel-plate flow chamber (GlycoTech, Rockville, MD, USA). Confluent HUVEC monolayers (up to Passage 4) were stimulated with TNF-{alpha} (10 ng/ml) for 4 h before perfusion. Jurkat T cells (1x106 cells/ml) were washed twice and resuspended in RPMI-1640 medium. In some experiments, Jurkat T cells were pretreated with DREG56 (anti-L-selectin mAb), PP2 (inhibitor of Lck), or siRNA to Lck. Cells were perfused over the activated HUVEC monolayer via a syringe pump at a shear stress of 1.2 dyn/cm2. The interactions between Jurkat T cells and the endothelial cells were visualized and recorded by using an inverted microscope (Olympus Optical, Tokyo, Japan) equipped with a camera (Panasonic, Yokohama, Japan) connected to a VCR and a television monitor. After 1 min perfusion with PBS/Ca/Mg/BSA flow buffer, cells entered the chamber and started to interact with the endothelium (t=0). Perfusion experiments were performed in 3 min.

Video images were evaluated afterwards, and the total number of rolling cells in 10 random fields of view (0.127 mm2) at 2–3 min of perfusion period was counted by digital imagine processing. Experiments were performed in triplicate on at least three separate occasions.

Statistical analysis
Data are expressed as means ± SEM unless indicated otherwise. The statistical significance of differences between means was determined by one-way ANOVA.


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RESULTS
 
L-selectin ligation with sulfatides induces robust tyrosine phosphorylation of Lck kinase and subsequent signaling events
It has been established that sulfatides function as the native ligands of L-selectin, which will cause a multivalent cross-linking of L-selectin [13 ]. A previous study has reported that Lck kinase became tyrosine-phosphorylated upon L-selectin stimulation with its mAb in Jurkat T cells [18 ], so we investigated whether sulfatides had a similar effect on Lck activation. Jurkat T cells were treated without or with W6/32 (antibody for HLA), DREG56 (antibody for L-selectin), or sulfatides, respectively, and then the cell lysates were subjected to SDS-PAGE to test the phosphorylation level of Lck kinase. The results showed that sulfatide cross-linking induced tyrosine phosphorylation of the Lck kinase, which was much stronger than DREG56. In contrast, no obvious variation was detected in the cells treated with control antibodies (Fig. 1A ). This result was also confirmed in the experiments conducted in primary T lymphocytes (Fig. 1B) .


Figure 1
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  		Figure 1. L-selectin ligation with sulfatides induces the robust tyrosine phosphorylation of Lck and subsequent signaling events. (A) Jurkat T cells were incubated in the absence of stimuli (Control) or with W6/32 (HLA; 10 µg/ml), DREG56 (10 µg/ml), or sulfatides (1 mg/ml) at 37ºC for 1 min. Lysates were prepared as described, and the immunoblots (IB) were probed with indicated antibodies. (B) The isolated primary T lymphocytes were treated as in A, and the immunoblots were probed with indicated antibodies. (C) Jurkat T cells were left untreated or pretreated with PP2 on ice for 1 h before being stimulated with sulfatides (1 mg/ml) at 37ºC for 1 min or not. Cells were fixed and stained with FITC-conjugated phalloidin. (a) Control; (b), BSA; (c), sulfatides; (d) sulfatides + PP2. Original bar = 20 µm. (D) HUVECs were preincubated without TNF-{alpha} (–) or with TNF-{alpha} (+) at 37ºC for 4 h. For the inhibition assay, Jurkat T cells were incubated with DREG56 or PP2 on ice for 1 h before being perfused over the HUVEC monolayer. The number of rolling cells was quantitated at a shear stress of 1.2 dyn/cm2 as described in Materials and Methods. *, P < 0.01, compared with positive control. Data are representative of three independent experiments.

Actin cytoskeleton rearrangement is considered as a marker of leukocyte activation, so we examined whether sulfatide stimulation could also result in F-actin polarization. As shown in Figure 1C , F-actin is significantly polarized and aggregated in several larger patches in activated Jurkat T cells stimulated with sulfatides compared with that in resting cells, but this polarization was reduced upon PP2 (the Src family kinase-specific inhibitor) preincubation. In leukocyte-endothelial cell interactions, L-selectin is the major selectin molecule responsible for the initial rolling of the leukocyte, so we wondered whether the Lck kinase played a role in this process. As shown in Figure 1D , the rolling of Jurkat T cells was increased dramatically on TNF-{alpha}-activated HUVECs compared with that on nonactivated HUVECs, and this increase was inhibited nearly completely by preincubating Jurkat T cells with L-selectin mAb DREG56. Meanwhile, PP2 treatment also abolished Jurkat T cell rolling on HUVECs, nearly to the basal level, indicating that the Lck kinase is critical in L-selectin-mediated leukocyte-endothelial cell interaction. Thus, sulfatides, as the activators of L-selectin, were able to activate subsequent signal transduction efficiently, in which Lck kinase played a significant role.

Lck kinase is associated with the L-selectin cytoplasmic tail
We next investigated the relationship between L-selectin and Lck kinase. Jurkat T cells were treated with sulfatides or not before being lysed for co-IP. As shown in Figure 2 A and B , irrespective of stimulation, the Lck kinase was present in the L-selectin-immunoprecipited complex and vice versa. We also examined the association of L-selectin with Lck kinase in vitro by using GST pull-down assays. GST-CytoL wild-type fusion protein was incubated with Jurkat T cell lysates, and the extensively washed protein complexes were separated by SDS-PAGE. Lck kinase was found in the GST-CytoL pull-down complexes (Fig. 2C) . Far Western assay under reducing conditions was performed to test whether the interaction between L-selectin and Lck kinase is direct. The data showed that L-selectin could interact with Lck kinase in a direct manner with its cytoplasmic tail (Fig. 2D) . To determine which domain of Lck kinase is responsible for the interaction of Lck and L-selectin, we prepared the GST-Lck SH2 and GST-Lck SH3 fusion proteins. As shown in Figure 2E , L-selectin was found in GST-Lck SH2 but not GST or GST-SH3 pull-down complexes. Far Western assay also confirmed that the Lck SH2 domain but not the SH3 domain interacted directly with L-selectin (Fig. 2F) . These results from in vivo and in vitro experiments suggested that Lck kinase interacted with the L-selectin cytoplasmic tail using its SH2 domain.


Figure 2
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  		Figure 2. Lck kinase is associated with the L-selectin cytoplasmic tail. (A) Jurkat T cells were incubated in the absence of stimuli (–) or with sulfatides (+; 1 mg/ml) at 37ºC for 1 min. Cells were lysed in lysis buffer, and the Lck immunoprecipitates were immunoblotted with the indicated antibodies. An unrelated mouse IgG (mIgG) immunoprecipitate was used as a control. (B) Whole cell lysates from Jurkat T cells, stimulated or not as in A, were immunoprecipited with the anti-L-selectin antibody and immunoblotted with indicated antibodies. An unrelated mIgG immunoprecipitate was used as a control. (C) Cell lysates from untreated or sulfatide-treated Jurkat T cells were incubated with GSH-sepharose beads coated with GST or GST-CytoL wild-type, prepared as described. Bound proteins were eluted and probed with indicated antibodies. (D) Lck immunoprecipitates from Jurkat T cells as in A were separated under reducing condition for Far Western assay by incubating with GST or GST-CytoL wild-type fusion proteins and immunoblotted with anti-GST antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. (E) Cell lysates from untreated or sulfatide-treated Jurkat T cells were incubated with GSH-sepharose beads coated with GST, GST-Lck SH2, or GST-Lck SH3. Bound proteins were eluted and probed with indicated antibodies. (F) L-selectin immunoprecipitates from Jurkat T cells as in B were separated under reducing conditions for Far Western assay by incubating with GST, GST-Lck SH2, or GST-Lck SH3 fusion proteins and immunoblotted with anti-GST antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. Data are representative of three independent experiments.

L-selectin cytoplasmic tail is a substrate of Lck kinase
It was reported that L-selectin stimulation with its antibody induced tyrosine phosphorylation of L-selectin itself in Jurkat T cells [18 ]. Based on the direct interaction between L-selectin and Lck kinase, we speculated that Lck kinase might be involved in this process. A kinase assay was performed to test the ability of endogenous Lck kinase to phosphorylate the cytoplasmic tail of L-selectin by using GST-CytoL wild-type fusion proteins as a substrate. As shown in Figure 3A , the Lck kinase immunoprecipited from the stimulated cells was able to phosphorylate the cytoplasmic tail of L-selectin, and this phosphorylation happened within 1 min of L-selectin stimulation. There is only one tyrosine residue (Y372) in the cytoplasmic domain of L-selectin (Fig. 3B) , so we generated two mutations on this site—Y372A and Y372F—to test whether this site is responsible for the interaction of L-selectin and Lck kinase. Data showed that all three GST fusion proteins could interact with Lck kinase, but the mutants attenuated the interaction, especially the Y372F mutant that almost abolished the interaction of L-selectin with Lck kinase (Fig. 3C) . These results indicated that the Lck kinase could quickly phosphorylate the L-selectin cytoplasmic tail after sulfatides stimulation, and Y372 of L-selectin played an important role in the association between Lck and L-selectin.


Figure 3
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  		Figure 3. L-selectin cytoplasmic tail is a substrate of Lck. (A) Jurkat T cells were stimulated with sulfatides for indicated times before being lysed as described and immunoprecipited with the anti-Lck antibody. An in vitro kinase assay using GST-CytoL fusion proteins prepared as described as substrates was detected by immunoblotting with an anti-phosphotyrosine (pTyr) antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. (B) A schematic diagram of the GST fusion protein of the L-selectin cytoplasmic tail and its mutants. WT, Wild-type. (C) Cell lysates from untreated or sulfatide-treated Jurkat T cells were incubated with GSH-sepharose beads coated with GST, GST-CytoL wild-type, and its mutants prepared as described. Bound proteins were eluted and probed with indicated antibodies. Data are representative of three independent experiments.

Lck kinase functions as an activator of ZAP-70
The activation of ZAP-70 kinase is one of the hallmarks of T cell activation, and its Tyr319 is the putative binding site for Lck kinase. We investigated whether ZAP-70 kinase is associated with Lck in L-selectin signaling induced by sulfatides stimulation. Data obtained from co-IP assays showed that Lck and ZAP-70 kinases were detected in each other’s immunoprecipited complexes (Fig. 4 A and B ). Preincubation of Jurkat T cells with PP2 did not affect the association but inhibited the phosphorylation of total and Tyr319 of ZAP-70 kinase nearly completely (Fig. 4A) , and piceatannol (the Syk/ZAP-70 kinase-specific inhibitor) had no effect on the association or the phosphorylation of Lck kinase (Fig. 4B) , suggesting that Lck kinase was associated with ZAP-70 and responsible for its activation induced by L-selectin ligation.


Figure 4
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  		Figure 4. Lck kinase functions as an activator of ZAP-70. (A) Jurkat T cells were left untreated or pretreated with PP2 on ice for 1 h before being stimulated with sulfatides (1 mg/ml) at 37ºC for 1 min or not. Cells were lysed in lysis buffer, and Lck immunoprecipitates were immunoblotted with the indicated antibodies. An unrelated mIgG immunoprecipitate was used as a control (data not shown). (B) Jurkat T cells left untreated or pretreated with piceatannol on ice for 1 h were stimulated or not as in A, and lysates were immunoprecipited with anti-ZAP-70 antibody and immunoblotted with indicated antibodies. An unrelated mIgG immunoprecipitate was used as a control (data not shown). (C) Cell lysates from untreated or sulfatide-treated Jurkat T cells were incubated with GSH-sepharose beads coated with GST, GST-Lck SH2, or GST-Lck SH3 fusion proteins prepared as described. Bound proteins were eluted and probed with indicated antibodies. (D) ZAP-70 immunoprecipitates from Jurkat T cells stimulated as in A were separated under reducing condition for Far Western assay by incubating with GST, GST-Lck SH2, or GST-Lck SH3 fusion proteins and immunoblotted with anti-GST antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. (E) Cell lysates from Jurkat T cells treated with sulfatides for indicated times were incubated with GSH-sepharose beads coated with GST-Lck SH2 fusion proteins prepared as described. Bound proteins were eluted and probed with indicated antibodies. Data are representative of three independent experiments.

To detect the direct interaction between Lck and ZAP-70, GST pull-down assays and Far Western assays were performed. As showed in Figure 4 C and D , ZAP-70 was found to interact directly with the SH2 but not the SH3 domain of Lck kinase. The results obtained above showed that the SH2 domain also mediated the interaction with the L-selectin cytoplasmic tail, so it became intriguing for us to investigate whether this domain could bind L-selectin and ZAP-70 kinase simultaneously. Results from GST pull-down assays showed that Lck kinase was constitutively associated with ZAP-70, and its association with L-selectin was attenuated after sulfatides stimulation for 2 min (Fig. 4E) , indicating that the interactions of Lck with ZAP-70 and L-selectin are regulated differently in the cell signaling events upon L-selectin activation. Thus, we concluded that ZAP-70 functioned as a direct target of Lck kinase in L-selectin signaling induced by sulfatides stimulation.

Lck kinase functions upstream of c-Abl
Our previous work showed that c-Abl kinase was required for L-selectin signaling induced by antibody cross-linking [33 ]. Here, we intended to explore and characterize the relationship between Lck and c-Abl kinases in L-selectin signaling. Jurkat T cells were treated with sulfatides for different times, and the phosphorylation levels of Lck and c-Abl kinases were detected. As shown in Figure 5A , phosphorylation of the c-Abl kinase peaked at 2 min after L-selectin ligation, and Lck did between 0.5 and 1 min, suggesting that Lck kinase was activated prior to c-Abl upon Jurkat T cell activation. This result was confirmed by the experiments conducted in primary T lymphocytes (Fig. 5B) . Experiments by antibody cross-linking of L-selectin also showed the same results (Supplemental Fig. 1). Results from co-IP assays indicated that Lck and c-Abl kinases were in the same immunocomplex (Fig. 5 C and D) . Pretreatment of Jurkat T cells with PP2 partly inhibited the association of Lck with c-Abl and the c-Abl phosphorylation, although STI571 (a specific inhibitor to c-Abl) had an effect on neither the association nor the phosphorylation of the Lck kinase. To test whether the kinase activity of c-Abl was affected by PP2 treatment, an in vitro kinase assay using GST-CrkII CTD as a substrate was performed. Sulfatide stimulation induced the elevation of c-Abl kinase activity, consistent with our previous results [33 ], but preincubation with PP2 declined this elevation (Fig. 5E) , indicating that Lck kinase plays a role in phosphorylation and kinase activity of c-Abl. Moreover, c-Abl was observed in GST-Lck SH3 but not GST-Lck SH2 pull-down complexes (Fig. 5F) , and Far Western assays also confirmed the direct interaction of the c-Abl and Lck SH3 domain (Fig. 5G) . However, we failed to find the binding domain in c-Abl kinase (data not shown). Thus, Lck was associated directly with c-Abl with its SH3 domain and functioned in the activation of c-Abl kinase.


Figure 5
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  		Figure 5. Lck kinase functions upstream of c-Abl. (A) Jurkat T cells were treated with sulfatides for indicated times before being lysed, the lysates were prepared as described, and the immunoblots were probed with indicated antibodies. (B) The isolated primary T lymphocytes were treated as in A, and the immunoblots were probed with indicated antibodies. (C) Jurkat T cells were left untreated or pretreated with PP2 on ice for 1 h before being stimulated with sulfatides (1 mg/ml) at 37ºC for 1 min or not. Cells were lysed in lysis buffer, and Lck immunoprecipitates were immunoblotted with the indicated antibodies. An unrelated mIgG immunoprecipitate was used as a control (data not shown). (D) Jurkat T cells left untreated or pretreated with STI571 for 1 h on ice were stimulated or not as in B, and lysates were immunoprecipited with the anti-c-Abl antibody and immunoblotted with indicated antibodies. An unrelated mIgG immunoprecipitate was used as a control (data not shown). (E) Jurkat T cells were treated as in B before being lysed and immunoprecipited with the anti-c-Abl antibody. An in vitro kinase assay using GST-CrkII-CTD fusion proteins, prepared as described as substrates, was detected by immunoblotting with anti-pTyr antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. (F) Cell lysates from untreated or sulfatide-treated Jurkat T cells were incubated with GSH-sepharose beads coated with GST, GST-Lck SH2, or GST-Lck SH3 fusion proteins prepared as described. Bound proteins were eluted and probed with indicated antibodies. (G) c-Abl immunoprecipitates from Jurkat T cells stimulated as in B were separated under a reducing condition for Far Western assay by incubating with GST, GST-Lck SH2, or GST-Lck SH3 fusion proteins and immunoblotted with anti-GST antibody. Parallel control samples detected by indicated antibodies were run to show equal loading. Data are representative of three independent experiments.

The kinase activity and intact SH2 domain of Lck are critical for L-selectin signaling induced by sulfatide engagement
The data presented above suggested that the activation of the Lck kinase was one of the first events induced by L-selectin stimulation in Jurkat T cells. To determine more fully the role of Lck kinase, we used siRNA to knock down Lck expression in Jurkat T cells. Whereas the mutated control siRNA was completely devoid of effect, Lck-specific siRNA was able to reduce Lck protein level, and the expressions of L-selectin, ZAP-70, and c-Abl were unaffected (Fig. 6A ). Meanwhile, the phosphorylation levels of L-selectin, ZAP-70, and c-Abl upon L-selectin engagement with sulfatides were decreased dramatically in Lck-KD cells (Fig. 6B) . Importantly, re-expression of wild-type murine Lck, which is not affected by the siRNA directly against human Lck, rescued tyrosine phosphorylation in the siLck-treated cells upon sulfatide stimulation (Fig. 6C) , indicating the important role of Lck kinase in L-selectin signaling.


Figure 6
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  		Figure 6. Lck is critical for L-selectin signaling induced by sulfatide engagement. (A) Jurkat T cells were transfected with 100 nM control (Lck232M3) or Lck-specific (Lck232) siRNA, and at 48 h post-transfection, the cells were harvested. Cell lysates were prepared as described, and the immunoblots were probed with indicated antibodies. (B) Jurkat T cells were treated as in A and were incubated in the absence of stimuli (–) or with sulfatides (+; 1 mg/ml) at 37ºC for 1 min. Cell lysates were prepared as described above, and immunoblots were probed with indicated antibodies. (C) Jurkat T cells were transfected with Lck232M3, Lck232, or Lck232 plus plasmid encoding murine wild-type Lck, and at 48 h post-transfection, the cells were stimulated with sulfatides. Cell lysates were subjected to immunoblotting with indicated antibodies. (D) A schematic diagram of Lck kinase and its mutants. Mutations on the kinase activity (Y505F for constitutively active and R273A for kinase dead), the SH3 domain (W97A), the SH2 domain (R154K), and SH3/SH2 (W97A/R154K) domains are indicated. (E) Jurkat T cells were transfected with Lck232M3, Lck232, or Lck232 plus plasmid encoding murine Lck or its mutants and treated as in C. (F) HUVECs were preincubated without TNF-{alpha} (–) or with TNF-{alpha} (+) at 37ºC for 4 h. Jurkat T cells were transfected with Lck232M3, Lck232, or Lck232 plus plasmid encoding murine Lck or its mutants, and at 48 h post-transfection, the cells were perfused over the HUVEC monolayer. The number of rolling cells was quantitated at a shear stress of 1.2 dyn/cm2 as described in Materials and Methods. *, P < 0.01, compared with positive control. Data are representative of three independent experiments.

The ability of murine Lck to rescue the effects of siLck on L-selectin signaling allows us to address the requirement of Lck function. As shown in Figure 6D , Lck has single SH3 and SH2 domains followed by a C-terminal tyrosine kinase domain. To directly test the role of Lck in the activation of the Jurkat T cell, we co-transfected cells with siRNA to Lck and plasmids encoding Lck or its mutants as described in Materials and Methods. The results showed that upon L-selectin ligation with sulfatides, constitutively active Lck (Y505F) rescued the tyrosine phosphorylation of L-selectin, ZAP-70, and c-Abl, and kinase-dead Lck (R273A) had no effect. Similarly, cells transfected with the SH2 mutant (R154K) and SH3/SH2 double mutant (W97A/R154K) also showed a poor phosphorylation, although the SH3 mutant (W97A) could partly rescue the tyrosine phosphorylation when stimulated with sulfatides (Fig. 6E) .

As we have identified the critical role of Lck in L-selectin-mediated leukocyte rolling on endothelial cells, we intended to investigate which domain(s) of Lck are responsible for this interaction. As shown in Figure 6F , the rolling of Lck-KD cells was obviously decreased on TNF-{alpha}-activated HUVECs compared with that of Jurkat T cells, and re-expression of wild-type murine Lck rescued this decrease. When we co-transfected cells with siRNA to Lck and plasmids encoding Lck mutants, results showed that constitutively active Lck (Y505F) not only rescued but also to some extent increased the cell rolling, and kinase-dead Lck (R273A) had no effect. Different from the effect on phosphorylation, the SH2 mutant (R154K) of Lck partly rescued the rolling, and the SH3 mutant (W97A) and SH3/SH2 double mutant (W97A/R154K) showed no increase.

To confirm the binding domain of Lck further, we coexpressed Lck wild-type, SH2 mutant, SH3 mutant, or SH3/SH2 double mutant with L-selectin, ZAP-70, or c-Abl in HEK293 cells. Co-IP experiments revealed that consistent with our results obtained above, disruption of the Lck SH3 domain or SH2 domain partly abrogated the association of Lck with c-Abl (Supplemental Fig. 2A) or L-selectin and ZAP-70 (Supplemental Fig. 2, B and C), and mutation of SH3 and SH2 domains completely abolished the binding of Lck with all three proteins (Supplemental Fig. 2). Thus, we believed that kinase activity and the intact SH2 domain of Lck play critical roles in L-selectin signaling induced by sulfatides stimulation.


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DISCUSSION
 
Here, we provide evidence that Lck kinase, directly interacted with the L-selectin cytoplasmic tail, can be phosphorylated rapidly upon L-selectin ligation with sulfatides. After activation, Lck phosphorylates the only tyrosine phosphorylation site, Tyr372, of the L-selectin cytoplasmic tail, promoting L-selectin signaling. Through its SH3 and SH2 domains, Lck interacts with c-Abl and ZAP-70 kinases to form a signaling complex and functions upstream of them. The critical role of Lck in L-selectin signaling is dependent on its kinase activity and the intact SH2 domain.

Previous studies have reported that L-selectin could function as a signaling molecule. The cytoplasmic domain of L-selectin consists of only 17 aa, which is considered to be a major part in L-selectin-mediated signaling. Calmodulin and {alpha}-actinin are the first molecules found to interact with the L-selectin cytoplasmic tail and are responsible for L-selectin shedding and for the association of L-selectin with actin cytoskeleton, respectively [19 , 38 ]. Ezrin-radixin-muesin proteins are demonstrated to interact with the cytoplasmic tail of L-selectin using affinity chromatography of lymphocyte extracts, and Arg357 and Lys362 of L-selectin contribute to this interaction [39 , 40 ]. Here, we indicate that Lck kinase is associated with the cytoplasmic tail of L-selectin directly through its SH2 domain (Fig. 2F) and phosphorylates it in less than 1 min upon L-selectin ligation with sulfatides (Fig. 5A) . Tyr372, the only phosphotyrosine site in the cytoplasmic tail of L-selectin, plays a role in the interaction between Lck kinase and L-selectin, as its mutation partly abolishes this interaction. Our results suggest that the Lck kinase, as an L-selectin-binding protein, could rapidly transduce L-selectin signals induced by sulfatides stimulation.

In our previous study, the c-Abl kinase was shown to constitutively associate with L-selectin, and the L-selectin cytoplasmic tail functioned as a substrate of c-Abl [33 ]. Here, we provide evidence that the Lck kinase is activated prior to c-Abl upon L-selectin activation, and the association between Lck and L-selectin is attenuated after sulfatides engagement for 2 min. Based on these results and previous studies about Lck and c-Abl [41 42 43 44 ], it is possible that L-selectin ligation with sulfatides induces Lck activation first, and the activated Lck phosphorylates the L-selectin cytoplasmic tail, promoting the activation of c-Abl kinase. After that, Lck detaches from the L-selectin cytoplasmic tail and transduces signals along the plasma membrane. The activated c-Abl reacts on (phosphorylates) the L-selectin cytoplasmic tail, enhancing L-selectin signaling to downstream molecules. It should be noted that our hypothesis does not rule out the possibility that other proteins may interact with the L-selectin cytoplasmic tail simultaneously and initiate signaling but does provide evidence for understanding how L-selectin orchestrates these cytoplasmic tyrosine kinases to transduce signals in the initial activation of Jurkat T cells triggered by sulfatides engagement.

It has been reported previously that the Lck kinase plays a role in T cell activation triggered by receptor ligation [45 46 47 ]. Here, we indicate that the Lck kinase is critical for L-selectin-mediated Jurkat T cell rolling on HUVECs (Fig. 1D) . As one of the rapidly activated kinases upon L-selectin stimulation, the loss of Lck, especially its kinase activity, inhibits the cell rolling nearly completely. Although the SH2 domain of Lck may contribute to the phosphorylation of downstream molecules, the SH3 domain is more important in Jurkat T cells rolling on HUVECs (Fig 6 E and F) . As the Lck-specific siRNA has no effect on the expression of L-selectin (Fig 6A) , and the Lck-specific inhibitor PP2 abolishes the F-actin polarization induced by L-selectin engagement (Fig 1C) , we speculate that Lck facilitates L-selectin-mediated cell rolling through its effect on the actin cytoskeleton rearrangement. Vav1 functions as a guanine nucleotide exchange factor of Rho family members, is involved in L-selectin signaling, and facilitates cell rolling through direct interaction with Lck (unpublished data). So, our results suggest that in L-selectin-mediated leukocyte-endothelial cell interactions, Lck kinase plays an essential role, and its SH3 and SH2 domains can perform distinct signaling functions.

As known, Lck adopts an inactive conformation, called "tail-bite", through the intramolecular interactions mediated by SH3 and SH2 domains. Once activated, both domains are released to act as an "adapter" molecule to bring in a signaling molecule(s) that is a tyrosine kinase itself or has associated kinase activity to promote signal transduction [48 ]. Syk kinase binds directly to the SH2 domain of Lck depending on its catalytic activity in the yeast two-hybrid system [49 ]. Upon TCR ligation, Lck recruits ZAP-70 to the ITAMs and then phosphorylates it at Tyr315 [50 , 51 ]. Here, we provide evidence that ZAP-70 is interacted directly and constitutively with the SH2 domain of Lck in Jurkat T cells. Unlike Syk kinase, the association of ZAP-70 and Lck is unrelated with the kinase activity of Lck or the phosphorylation of ZAP-70, as neither PP2 nor piceatannol treatment plays a role on this association. PP2 treatment completely inhibits the phosphorylation of ZAP-70, indicating that Lck kinase activity is important for ZAP-70 activation. In Lck-KD cells, only the re-expression of Lck with kinase activity and the intact SH2 domain can rescue the phosphorylation of ZAP-70 (Fig. 6E) . Meanwhile, coexpression of ZAP-70 and Lck in HEK293 cells exhibits that Lck with a destroyed SH2 domain can still bind ZAP-70, suggesting that other domains of Lck may also be involved in this association. Our results suggest that Lck kinase interacts directly with ZAP-70 through its SH2 domain to form a signaling complex with L-selectin and activates ZAP-70 via its kinase activity to transduce signals downstream.

The SH3 domain of Lck kinase has been shown to bind several signaling molecules implicated in different signaling pathways. PI-3K binds to the Lck SH3 domain and is capable of activating the MAPK pathway in some cell types [52 , 53 ]. Upon TCR ligation, Lck is localized to lipid rafts and is negatively regulated by Cbl ubiquitin ligase through its SH3 domain [54 , 55 ]. Here, we find that the c-Abl kinase is also interacted with the SH3 domain of Lck in Jurkat T cells upon L-selectin ligation. Coexpression assay in HEK293 cells shows that Lck with the SH3 domain mutation only partly abolishes the association with c-Abl, indicating that the SH3 domain of Lck is one of the domains but not the only domain responsible for this interaction. Unlike ZAP-70, the association of c-Abl with Lck is inducible, and PP2 treatment partly inhibits this association. The kinase activity of Lck is important for c-Abl activation and kinase activity, as PP2 treatment significantly inhibits the phosphorylation of c-Abl itself and the phosphorylation of CrkII, an endogenous substrate of c-Abl (Fig. 5) . In Lck-KD cells, re-expression of Lck with kinase dead fails to rescue the activation of c-Abl (Fig. 6E) . Our results suggest that upon Jurkat T cell activation, Lck interacts with c-Abl through its SH3 domain and regulates c-Abl activation.

In conclusion, the findings of this study suggest that in Jurkat T cells, the Lck kinase is constitutively associated with ZAP-70 at the cytoplasmic tail of L-selectin. Upon sulfatides engagement, Lck kinase is activated rapidly and phosphorylates L-selectin. Meanwhile, the association of Lck and c-Abl is enhanced, which induces the activation of c-Abl. After that, the Lck kinase is detached from L-selectin. This signaling dynamic at the cytoplasmic tail of L-selectin ensures signal transduction downstream.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Natural Science Foundation of China (30500927 and 30570928). We thank Novartis Pharma Switzerland AG for providing inhibitor STI571 and Drs. Hamid Band, Ottmar Janssen, Nicolas Foray, Giorgio Scita, Hidesaburo Hanafusa, and Oreste Acuto for providing plasmids that made this work possible.

Received February 3, 2008; revised June 26, 2008; accepted June 27, 2008.


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