c-Abl is required for the signaling transduction induced by L-selectin ligation

L-selectin ligation induces CSF-1 gene transcriptional activation

The activation of T cells via the interaction of L-selectin and its mAb leads to the stimulation of multiple intracellular events. In our study, we investigated whether the ligation of L-selectin was able to induce CSF-1 gene transcriptional activation. Jurkat cells were transfected with the CSF-1-luc vector, and after 24 h, the transfected cells were incubated at 37°C for 6 h without antibody or with mouse IgG, W6/32 or DREG56. The cells were then lysed to measure both the firefly and the Renilla luciferase. The data shown indicated that the CSF-1 gene transcription was clearly induced in anti-L-selectin mAb-treated cells. In contrast, no significant signal was detected in the cells treated with the control antibodies (Fig. 1A). We also tested the endogenous CSF-1 gene transcription. Jurkat cells were incubated with DREG56 at 37°C for the indicated time. Cells were then lysed and analyzed by RT-PCR for CSF-1 gene transcription. As shown in Fig. 1B, treatment of cells with an anti-L-selectin antibody for 30 min resulted in the maximal activation of CSF-1 gene transcription. The activation decreased when the cells were treated with DREG56 for 60 min. Treatment of the isolated primary T lymphocytes with anti-L-selectin antibody for 30 min also resulted in the maximal activation of CSF-1 gene transcription (Fig. 1C). To better understand the physiological role of L-selectin, we also tested the CSF-1 gene transcriptional activation in the sulfatide-stimulated primary T lymphocytes. The isolated cells were incubated with 100 μg/mL or 250 μg/mL sulfatides for different times. As shown in Fig. 1D, CSF-1 mRNA increased about tenfold when the lymphocytes were stimulated with sulfatides, while CSF-1 mRNA increased about threefold when cells were stimulated with DREG56 ligation (Fig. 1C). The results suggested that sulfatides ligation induced CSF-1 gene transcription more strongly than L-selectin engagement with DREG 56.

c-Abl kinase regulates CSF-1 gene transcription induced by L-selectin ligation

c-Abl kinase has been reported to play an important role in the cytoskeleton reorganization induced by adhesion molecules cross-linking. To assess whether c-Abl kinase could function as a necessary kinase in CSF-1 gene transcriptional activation triggered by L-selectin ligation, WT c-Abl expression vector or control vector and the CSF-1-luc vector were co-transfected in Jurkat cells. As shown in Fig. 2A, the overexpression of WT c-Abl kinase could increase CSF-1 gene transcription, while overexpression of a KD mutant c-Abl kinase was not able to regulate CSF-1 transcription (Fig. 2B). We also tested the endogenous CSF-1 transcriptional level in the Jurkat cells pretreated with STI571 for 30 min before they were activated by antibody ligation. STI571 is a potent and specific blocker of c-Abl kinase with an IC₅₀ of ∼650 nM for inhibition of its kinase activity. The CSF-1 gene transcription of the STI571-treated cells was decreased dramatically compared to the cells without STI571 pretreated (Fig. 2C).

L-selectin ligation increases c-Abl kinase activity

We next examined the c-Abl kinase activity in response to L-selectin ligation stimulation. The endogenous c-Abl kinase activity was tested by analyzing the phosphorylation status of the endogenous c-Abl substrate Crk. The results showed that the level of Crk phosphorylation was increased within 5 min of L-selectin stimulation (Fig. 3A). To determine if the phosphorylation of Crk was dependent on c-Abl kinase activity, we treated Jurkat cells with STI571 prior to activation and found that STI571 inhibited the Crk phosphorylation induced by L-selectin ligation (Fig. 3B). We then tested the c-Abl kinase activity using GST-CrKII-CTD as a substrate in an in vitro kinase assay. The results demonstrated that c-Abl kinase activity was elevated after L-selectin ligation (Fig. 3C), and STI571 incubation could reduce the phosphorylation of GST-CrKII-CTD (Fig. 3D). The data described above indicates that c-Abl kinase activity was increased after L-selectin ligation.

Activated c-Abl kinase enhances Zap70 kinase activity downstream of L-selectin ligation

The activation of Zap70 kinase is one of the first events following T cell activation. So we were interested to discover if Zap70 kinase was involved in the L-selectin ligation-induced signal transduction in which c-Abl kinase was necessary. We first investigated Zap70 kinase activity after L-selectin ligation. As shown in Fig. 4A, Zap70 phosphorylation was greatly increased in the L-selectin ligation-stimulated cells, and the kinase activity remained elevated for 20 min after which it decreased to baseline values. It has been reported that phosphorylation of Tyr319 of Zap70 (Y319) serves an important role for the Zap70 kinase activity. We next tested the Y319 phosphorylation of Zap70 kinase after L-selectin ligation. The results showed that the phosphorylation of Y319 site of Zap70 kinase was elevated in the stimulated cells (Fig. 4A). To define if c-Abl kinase could regulate Zap70 kinase activity, Jurkat cells were pre-incubated with piceatannol (the syk/Zap70-specific kinases inhibitor) before ligation with DREG56. The results showed that c-Abl kinase activity was not affected in the piceatannol-treated cells (Fig. 4B). We also assessed the tyrosine phosphorylation of Zap70 kinase after the cells were treated with STI571. As shown in Fig. 4C, the phosphorylation of Y319 site of Zap70 kinase, other than the total phosphorylation of Zap70, was greatly inhibited in the presence of STI571. All these results indicate that the activated c-Abl kinase could regulate Zap70 kinase activity by phosphorylating the Y319 site downstream of L-selectin ligation.

c-Abl kinase increases CSF-1 gene transcription via regulating Zap70 kinase activity

c-Abl kinase can increase CSF-1 gene transcriptional activity and regulate Zap70 kinase activity in the L-selectin-stimulated Jurkat cells. To determine whether Zap70 kinase was involved in the CSF-1 gene transcription triggered by L-selectin ligation, Jurkat cells were transfected with WT Zap70, KD Zap70 or Y319F Zap70 expression vector in the presence of CSF-1-luc vector. After 24 h, the transfected cells were incubated with DREG56 at 37°C for 6 h. The cells were then lysed and analyzed by a double luciferase reporter assay. The results showed that overexpression of WT Zap70, other than KD Zap70 or Y319F Zap70, could increase the CSF-1 gene transcription (Fig. 5A). The increased CSF-1 gene transcriptional activity was reduced if the WT Zap70-transfected cells were preincubated with STI571 or piceatannol before stimulation with L-selectin ligation (Fig. 5B). With pretreatment of the WT c-Abl kinase-overexpressing cells with piceatannol or STI571 before L-selectin ligation, the CSF-1 gene transcriptional activation was also reduced (Fig. 5C). Consistently, the endogenous increased CSF-1 gene transcriptional activity induced by L-selectin ligation was inhibited by STI571 and piceatannol incubation (Fig. 5D). The results above indicate that Zap70 kinase was involved in the CSF-1 gene transcription, which was dependent on the c-Abl kinase activity.

The cytoplasmic tail of L-selectin is associated with c-Abl kinase and Zap70 kinase in activated Jurkat cells

We have shown that c-Abl and Zap70 kinases were activated in the L-selectin-stimulated Jurkat cells. Next we investigated the relationship of L-selectin with c-Abl kinase and Zap70 kinase. Jurkat cells were stimulated and lysed, and the lysates were used for co-immunoprecipitation. As shown in Fig. 6A, c-Abl kinase and Zap70 kinase were present in the L-selectin immunoprecipitated complex. L-selectin was also detected in the c-Abl kinase and Zap70 kinase immunoprecipitated complexes (Fig. 6B and C). STI571 preincubation reduced the association of L-selectin and c-Abl kinase and also decreased the association between L-selectin and Zap70 kinase (Fig. 6A–C). These results indicate that L-selectin was associated with c-Abl kinase and Zap70 kinase in the L-selectin ligation-stimulated cells.

We also examined the association of L-selectin with c-Abl kinase and Zap70 kinase in vitro using a GST pull-down assay. GST-CytoL WT fusion protein was incubated with Jurkat cell lysates and the extensively washed protein complexes were separated by SDS-PAGE. c-Abl kinase and Zap70 kinase were found in the GST pull-down complexes (Fig. 6D). To detect whether GST-CytoL directly binds to c-Abl kinase and Zap70 kinase, a far Western blot analysis was performed. The results showed that GST-CytoL fusion protein directly interacted with c-Abl kinase (Fig. 6E), while it could not interact with Zap70 kinase directly (Fig. 6F). To determine which domain of c-Abl kinase was responsible for the interaction of c-Abl kinase and L-selectin, GST-c-Abl SH2, GST-c-Abl NSH were incubated with the lysates from unstimulated or stimulated Jurkat cells. As shown in Fig. 6G, L-selectin was found in the GST-c-Abl SH2, but not GST or NSH pull-down complexes. We therefore concluded that L-selectin connects directly with the c-Abl kinase SH2 domain, but indirectly with Zap70 kinase in Jurkat cells.

L-selectin cytoplasmic tail is a substrate of c-Abl kinase

We have shown that c-Abl kinase could interact with the cytoplasmic domain of L-selectin in the activated Jurkat cells triggered by L-selectin ligation. To determine whether the interaction between c-Abl kinase and L-selectin led to a modification of L-selectin, we examined the ability of endogenous c-Abl kinase to phosphorylate the cytoplasmic tail of L-selectin by carrying out a kinase assay using GST-CytoL fusion protein as a substrate and immunoprecipitated c-Abl kinase derived from unstimulated or L-selectin engagement-stimulated Jurkat cells. As shown in Fig. 7A, the c-Abl kinase obtained from stimulated cells was able to phosphorylate the cytoplasmic tail of L-selectin. There is only one tyrosine residue (Y372) in the cytoplasmic domain of L-selectin (Fig. 7B). To test whether this site is responsible for the association of L-selectin and c-Abl kinase, GST-CytoL WT, GST-CytoL Y372A and GST-CytoL Y372F were used for GST pull-down assays. The results showed that all these GST fusion proteins could associate with c-Abl kinase, with the GST-CytoL Y372F having a lower interaction with c-Abl kinase (Fig. 7C). These results suggest that c-Abl kinase could phosphorylate L-selectin Y372, and this modification plays a role, although not a critical, in the association between c-Abl kinase and L-selectin.

c-Abl kinase associates with Zap70 in the activated Jurkat cells

We have shown that L-selectin interacts directly with c-Abl SH2 domain, but not directly with Zap70 kinase in the activated Jurkat cells. Next we tested the relationship between c-Abl kinase and Zap70 kinase using a co-immunoprecipitation assay. The results showed that c-Abl kinase could be found in the immunoprecipitate of Zap70 kinase, and Zap70 kinase was present in the c-Abl kinase immunoprecipitated complexes (Fig. 8A and B). STI571 could reduce the association between c-Abl kinase and Zap70 kinase, indicating that c-Abl kinase stimulation was responsible for the association between c-Abl kinase and Zap70 kinase. To better understand the physiological role of L-selectin, we tested the association of c-Abl and ZAP70 in primary T lymphocytes stimulated by L-selectin engagement with DREG56 or sulfatides. We first tested the association between c-Abl and Zap70 in primary T lymphocytes. Before the primary T lymphocytes were stimulated with DREG56 or sulfatides, the cells were pre-incubated with STI571 or piceatannol. The treated cells were lysed, and the lysates were used for co-immunoprecipitation. As shown in Fig. 8C, c-Abl appeared in the Zap70 kinase immunoprecipitated complexes. Zap70 was detected in the c-Abl kinase immunoprecipitated complexes (Fig. 8D). STI571 and piceatannol pre-incubation reduced the association of c-Abl kinase and Zap70 kinase (Fig. 8C and D). These results indicated that c-Abl kinase was associated with Zap70 kinase in normal primary T cells stimulated by L-selectin ligation. Our GST-c-Abl SH2 pull-down assay showed that Zap70 kinase was associated with the SH2 domain of c-Abl kinase (Fig. 8E), and the far Western blot assay results further demonstrated that Zap70 kinase was connected with c-Abl SH2 domain directly (Fig. 8F). As mentioned above, both L-selectin and Zap70 kinase could interact with the SH2 domain of c-Abl kinase. To test whether these two molecules interacted with c-Abl kinase simultaneously, the association between c-Abl SH2 domain and Zap70 kinase or c-Abl SH2 domain and L-selectin was analyzed using a GST pull-down assay. As shown in Fig. 8G, c-Abl kinase was connected with L-selectin constitutively and the connection between c-Abl kinase and Zap70 kinase was enhanced after L-selectin stimulation. These data indicate that the activated c-Abl kinase interacted with Zap70 kinase in the L-selectin-stimulated cells. The connection between c-Abl kinase and Zap70 kinase was strengthened following L-selectin ligation.

Cell culture, reagents and antibodies

The human leukemic Jurkat T cell line was grown in RPMI 1640 (Gibco) containing 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. The primary T lymphocytes were isolated as described previously 59.

W6/32 (anti-human HLA-A,B,C mAb, mouse IgG2a, 14–9983) was purchased from eBioscience; DREG56 (anti-L-selectin mAb, mouse IgG₁, sc-18851), K12 (anti-c-Abl polyclonal antibody, rabbit IgG, sc-131), PY99 (anti-phosphotyrosine mAb, mouse IgG_2b, sc-7020), 1E7.2 (anti-Zap70 mAb, mouse IgG, sc-32760) from Santa Cruz Biotechnology, Inc (Santa Cruz, CA); G7781 (anti-glutathione S-transferase antibody, rabbit IgG) and piceatannol (syk/Zap70-specific kinases inhibitor) from Sigma; 8E9 (anti-c-Abl mAb, mouse IgG) from BD biosciences PharMingen; 65E4 (anti-phospho-Zap70 Tyr319 mAb, Rabbit IgG) from cell signaling technology; and anti-Crk antibody (mouse IgG2a) from BD transduction laboratories. STI571 (a special inhibitor to non-receptor tyrosine kinase c-Abl) was a gift of Novartis Pharma Switzerland AG (Basel, Switzerland). ECL Plus Western blotting detection reagents (RPN2132) and Glutathione Sepharose 4B^TM (17–0756–01) were purchased from Amersham Biosciences.

Recombinant DNA constructs

The luciferase reporter vector with a CSF-1 promoter, pREP4-CSF-1-luc, and the pREP7-Renilla-luc vector, were kindly provided by Dr. Keji Zhao (NIH, Maryland). The expression vectors encoding WT c-Abl and KD c-Abl were the gifts of Dr. Hidesaburo Hanafusa (The Rockefeller University, Japan). The GST-Crk-CTD plasmid was the gift of Dr. Giorgio Scita (European Institute of Oncology, Italy). The GST-c-Abl-NSH (489–543) and GST-c-Abl-SH2 plasmids were kindly given by Dr. Nicolas Foray (European Synchrotron Research Facility, France). The expression vectors encoding WT Zap70, KD Zap70 and Y319F Zap70 were kindly provided by Dr. Oreste Acuto (the Molecular Immunology Unit, Department of Immunology, Institute Pasteur, France). We sub-cloned Zap70 fragments in the pcDNA3.1 expression vector. 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 sub-cloned in pGEX vector. Inactivating mutations of putative phosphorylation sites in the cytoplasmic domain of L-selectin were introduced by use of altered PCR primers. The fragments obtained were sub-cloned into pGEX as described for the WT sequence.

GST and GST-fusion protein purification

E. coli strain BL-21 (DE3) transformed with GST or GST-fusion protein expression vectors were incubated in 200 mL LB culture medium with 50–100 µg/mL ampicillin and induced with 0.3 mM IPTG for 3 h at 37°C. 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 of aprotinin and leupeptin) and disrupted by sonication. Lysates were cleared by centrifugation at 15 000 × g at 4°C for 30 min. The supernatants were applied to a GSH-Sepharose column. After extensive washing with the lysis buffer, the isolated proteins were stored at 4°C for experiments (for not more than 1 week).

RT-PCR

Jurkat cells were incubated at 37°C for the indicated time with 10 µg/mL DREG56 or control antibody. The total RNA of the cells was extracted with the TRIzol isolation system. Total RNA (1 μL) was reverse transcribed in a total volume of 20 μL and real-time PCR using SYBR green fluorescence was performed. Each real-time PCR reaction consisted of 1 μL RT product, 12.5 μL SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA), and 500 nM forward and reverse primers. Reactions were carried out on an ABI PRISM 7000 Sequence Detection System (AB Applied Biosystems) for 40 cycles (95°C for 15 s, 60°C for 1 min) after initial 10 min incubation at 95°C. The following primers were used: β-actin forward: 5′-ATGCCAGGGTACATGGTGGT-3′, β-actin reverse: 5′-TCGTGCGTGACATTAAGGAG-3′; CSF-1 forward: 5′-GTCATATGTTGAGCCTGTGG-3′, CSF-1 reverse: 5′-GGCTACGGAGATGACAGAAT-3′.

Luciferase reporter assay

For luciferase assays, transfected cells were stimulated with the indicated antibodies for 6 h and then lysed in 25 μL of an appropriate reporter lysis buffer to measure both the Firefly and the Renilla luciferase (Promega). Luciferase activity was assayed in triplicate by luminometry using the Promega double-luciferase assay system. Luciferase activity was expressed as fold increase relative to the basal activity seen in un-stimulated cells.

Kinase assay

For activation, 1 × 10⁷ Jurkat cells per sample were washed twice in sterile HEPES saline (H/S; 132 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄) and stimulated at 37°C with DREG56. Cell stimulation was terminated by lysis in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM each NaF, Na₃VO₄, and β-glycerophosphate, and 20 μg/mL aprotinin/leupeptin. After 15-min incubation on ice, cells were centrifuged at 15 000 X g for 25 min, and lysates were incubated with anti-c-Abl antibody (K12). After 2 h, 20 μL protein A-Sepharose beads (50% slurry) was added to the antibody/lysated mixture for 1 h. Immunoprecipitates were washed at least four times (1 mL) with lysis buffer, and then washed three times with the kinase buffer (25 mM Tris, pH 7.5, 2 mM DTT, 5 mM β-glycerophosphate, 1 mM Na₃VO_4, 10 mM MgCl₂). After 5-min pre-incubation at 30°C, 30-μL reactions were initiated by the addition of 3 μg GST-CrKII-CTD or GST-CytoL and 5 μM ATP. After 30 min, reactions were terminated by the addition of 20 μL 3X SDS sample buffer, and resolved by SDS-PAGE.

Immunoprecipitation and Immunoblotting

Jurkat cells were stimulated and lysed as above. Lysates were centrifuged at 15 000 × g for 25 min, and the supernatants were incubated with the indicated antibodies. After 2 h, 20 μL protein A/G-Sepharose beads (50% slurry) was added to the antibody/lysated mixture for 1 h. Immunoprecipitates were washed three to six times with lysis buffer and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes using the chilled transfer buffer (25 mM Tris base, 192 mM glycerin, and 20% methanol) at 100 V for 1 h at 4°C. After protein transfer, the nitrocellulose membranes were washed with TBST (20 mM Tris base, 500 mM NaCl, 0.05% Tween-20, pH 7.5) at least three times and immediately incubated with 5% non-fat milk, the indicated primary antibody and the HRP-conjugated secondary antibody at 37°C for 1 h. Chemiluminescent detection was performed by using ECL plus Western blotting reagents.

Isolation of cellular proteins interacting with GST fusion protein (pull-down)

Jurkat 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, washed four times with lysis buffer, and the bound proteins were eluted by boiling in SDS-sample buffer and analyzed by SDS-PAGE.

Far Western analysis

To determine direct interaction of either c-Abl or selected c-Abl domains with the electrophoresed, nitrocellulose-bound proteins, SDS-PAGE and Western blotting were performed as described above, followed by blocking of the membranes with phosphate-buffered saline containing 3% BSA and 0.1% Tween-20. Membranes were then incubated overnight at 4°C with a blocking buffer containing 10 μg/mL of the indicated GST fusion protein or GST protein. Bound GST proteins were detected by incubation of the membrane with a rabbit anti-GST mAb for 1 h, followed by an HRP-conjugated sheep anti-rabbit IgG and ECL development.