Correspondence to Carl G. Gahmberg: carl.gahmberg{at}helsinki.fi
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Abbreviations used in this paper: ICAM, intercellular adhesion molecule; J, Jurkat; LFA-1, leukocyte function-associated antigen-1; sICAM, soluble ICAM; TCR, T cell receptor; wt, wild-type.
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Introduction |
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Dynamic adhesion is especially important in the immune system, where cells need to attach and detach continuously. The leukocyte function-associated antigen-1 (LFA-1) integrin (Lß2 or CD11a/CD18) is expressed exclusively in leukocytes and is of fundamental importance to the function of the immune system (Springer, 1990; Gahmberg, 1997). LFA-1 mediates cell adhesion under various conditions, e.g., during immunological synapse formation between the T cell and the antigen-presenting cell and during leukocyte emigration from the bloodstream into tissues. Whereas T cell receptor (TCR)mediated adhesion is slow and sustained, chemokine-induced adhesion is fast and rapidly reversible. Both affinity-dependent and -independent mechanisms have been postulated as being important in the regulation of integrin activation (van Kooyk and Figdor, 2000; Carman and Springer, 2003; Calderwood, 2004). These mechanisms are not mutually exclusive, and different modes of integrin activation may involve different mechanisms working alone or together. For example, TCR-induced activation of LFA-1 has not been shown to involve affinity regulation (conformational changes) in the integrin, but instead has been closely correlated with the spreading phenotype of T cells and actin cytoskeleton rearrangements (Stewart et al., 1996, 1998). In contrast, chemokines mediate rapid conformational changes in LFA-1, as measured by activation epitope expression with mAbs and the measurement of soluble ligand binding to the integrin (Weber et al., 1999; Constantin et al., 2000). Chemokine-induced adhesion also involves the clustering of integrins (Constantin et al., 2000). Ligands can also induce conformational changes and clustering of integrins (Cabanas and Hogg, 1993; Li et al., 1995; Kotovuori et al., 1999; Kim et al., 2004).
Phosphorylation is a common mechanism for the regulation of surface receptor function and has also been reported in integrins, but its role in integrin regulation has remained only partially understood (Fagerholm et al., 2004). LFA-1 is phosphorylated on both the and ß chains, with the
chain being constitutively phosphorylated, whereas ß chain phosphorylation becomes detectable after inside-out stimulation of the integrin (Hara and Fu, 1986; Chatila et al., 1989; Valmu and Gahmberg, 1995). The
chain phosphorylation sites have not been mapped, and their functions are completely unknown. In contrast, the ß chain phosphorylation sites are known (Hibbs et al., 1991; Fagerholm et al., 2002b; Hilden et al., 2003). The main phosphorylation site after phorbol ester stimulation of cells is Ser756, but this site is not involved in regulating adhesion (Hibbs et al., 1991). The threonine triplet (Thr758760) in the ß2 chain is important for adhesion, interactions with the actin cytoskeleton, and modulation of cell spreading (Hibbs et al., 1991; Peter and O'Toole, 1995). Interestingly, threonine phosphorylation of the ß chain has been reported (Valmu and Gahmberg, 1995) and threonine-phosphorylated integrins distribute preferentially to the actin cytoskeleton in cells (Valmu et al., 1999a). Additionally, it has been shown that 14-3-3 proteins from cell lysates interact with a Thr758-phosphorylated ß2 integrin peptide in vitro (Fagerholm et al., 2002b), but whether the interaction occurs in vivo or plays a role in adhesion has not been discovered. In this study, we investigated the role of both
and ß chain phosphorylations in the regulation of LFA-1mediated adhesion.
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Results |
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Mutation of Ser1140 of L abolishes T cell adhesion to ICAM-1 induced by the activating antibody MEM-83 and MEM-83induced affinity regulation of LFA-1
We next generated stable J-ß2.7 cell transfectants expressing wt L and S1140A-
L to examine the role of the phosphorylation site in cells (Fig. 3 A). Mutation of Ser1140 to Ala did not affect the heterodimerization or cell surface expression of LFA-1. The phosphospecific
L antibody was used for the characterization of the J-ß2.7 cell clones. The
L chain was easily detected in J-ß2.7 wt
L cells, whereas no significant staining of
L was seen in J-ß2.7 S1140A-
L cells with or without phorbol ester activation (Fig. 3 B).
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The phosphorylation site in L is involved in chemokine- and ligand-induced affinity regulation of the integrin
One of the best characterized examples of LFA-1 affinity modulation is the activation of integrin-dependent leukocyte arrest and migration through G proteincoupled receptors for chemokines (Constantin et al., 2000). SDF-1, a CXC chemokine, has been shown to induce a rapid and transient activation of LFA-1 in J-ß2.7 wt
L cells that can be detected by expression of the mAb24 epitope (Weber et al., 1999). To determine whether the S1140A mutation affected chemokine-triggered mAb24 expression, we stimulated J-ß2.7 transfectants with SDF-1
and studied the mAb24 epitope by immunofluorescence. No mAb24 staining was seen in nonstimulated cells (Fig. 5 A). Mg/EGTA treatment was used as a positive control to show the ability of transfectants to express the mAb24 epitope (Fig. 5, B and E). After SDF-1
stimulation, clear staining of mAb24 was detected on the wt
L cells, but not on the S1140A-
Lexpressing cells (Fig. 5, C and E).
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Active Rap1 cannot activate the S1140A-Lmutated integrin in cells
The small GTPase Rap1 is a potent activator of LFA-1 that is needed in different modes of LFA-1 activation, including activation from the outside of the cell with divalent cations or antibodies (Katagiri et al., 2000, 2002; de Bruyn et al., 2002; Sebzda et al., 2002). Transfection of leukocytes with an active form of Rap1 (Rap1V12) has been shown to increase LFA-1 affinity, as measured by increased ability to bind sICAM-1 (Katagiri et al., 2003). GFP-Rap1V12 was transfected into J-ß2.7 cells stably expressing wt L or S1140A-
L (Fig. 6). The expression level of Rap1V12 in wt
L and S1140A-
Lexpressing J-ß2.7 cells was the same (Fig. 6 A). However, Rap1V12 was unable to activate the S1140A mutant, even if increased adhesion of wt
Lexpressing cells could be observed after Rap1V12 transfection (Fig. 6 B).
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Mutation of Thr758 in ß2 or blocking of 14-3-3 binding to the ß2 chain reduces constitutive COS cell adhesion
Mutation of Thr758 in the ß2 chain has previously been shown to affect adhesion of LFA-1 to ICAM-1 when transfected into COS cells, where LFA-1 is constitutively active, and into LAD cells (a B lymphoblastoid cell line lacking LFA-1), where LFA-1 can be activated with phorbol ester (Hibbs et al., 1991). We used the COS cell system to investigate the effect of direct 14-3-3 association with the ß2 chain without interfering with activating signals of the integrin and because a T cell model lacking ß2 integrins is currently unavailable.
Phorbol ester did not stimulate LFA-1mediated adhesion to ICAM-1 in this system, but MEM-83 did have a stimulatory effect (Fig. 8 A). As previously reported, the Thr758Ala mutation significantly reduced the constitutive integrin-mediated adhesion of transfected cells to ICAM-1 (Fig. 8 A). MEM-83 could still activate the Thr-mutated integrin, albeit to a lower degree than for wt LFA-1 (Fig. 8 A), showing that activating conformational changes could still occur for the mutated integrin. However, when both Ser1140 and Thr758 were mutated, not even MEM-83 could activate the integrin-mediated adhesion (Fig. 8 A), even if the singly mutated Ser1140Ala-L together with wt ß2 could normally mediate adhesion in COS cells. Because the S1140A-
Lmutated integrin is adhesion deficient in J-ß2.7 cells, but not in COS-1 cells, it is clear that these cells are not a useful model for studying LFA-1 function in all cases.
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14-3-3 binding to phosphorylated ß2 is involved in cytoskeletal rearrangements
Neither the wt LFA-1 nor the Thr758Ala mutant of the ß2 chain showed any binding of mAb24 in COS cells (Fig. 9 A). Additionally, phorbol esters and TCR ligation have been reported not to induce sICAM-1 binding or mAb24 expression in T cells, although cell adhesion to coated ICAM-1 can be readily detected (Stewart et al., 1996). In contrast, TCR triggering and phorbol esters have been closely associated with a spreading phenotype of T cells (Stewart et al., 1996). Furthermore, the TTT motif of the integrin has been closely associated with actin reorganization events, but not affinity changes, in integrins (Peter and O'Toole, 1995). Indeed, the Thr758 mutation was shown to significantly reduce cell spreading on ICAM-1 as examined by FITC-phalloidin staining of polymerized actin (Fig. 9 B). In addition, R18wt-transfected cell spreading on ICAM-1 was almost completely abolished (Fig. 9, C and D). Quantitation of transfected cell spreading showed that R18wt and LFA-1 cotransfected cells only spread at later time points, when other untransfected COS-1 cells started spreading unspecifically on ICAM-1 (Fig. 9 D). The mutant R18 construct did not block spreading. These results show that 14-3-3 binding to the phosphorylated ß2 integrin is important for integrin effects on the cell cytoskeleton.
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Discussion |
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L has been shown to be constitutively phosphorylated in T cells (Hara and Fu, 1986; Chatila et al., 1989), but the phosphorylation site and stoichiometry of
L phosphorylation has not been assessed. We have now mapped the phosphorylation site in the
L cytoplasmic domain to Ser1140 and shown that
40% of surface
L was phosphorylated in T cells. A similarly high stoichiometry of phosphorylation already found in resting cells has been shown for the
4 integrin (Han et al., 2001). In analogy with the
4 integrin, where paxillin binding to the nonphosphorylated integrin rather than to the phosphorylated form excludes this adaptor protein from the leading edge of the cell and thus regulates cell migration (Goldfinger et al., 2003), constitutive phosphorylation of the
L tail may play profound roles in spatiotemporal regulation of integrin functions.
By mutating Ser1140, we have shown that the nonphosphorylatable LFA-1 chain expressed in a T cell line can no longer be activated by agents that have previously been demonstrated to induce high-affinity forms of the integrin; i.e., by an activating antibody (MEM-83); by ligands or chemokines, as detected by adhesion to ICAM-1; by sICAM-1 binding; or by mAb24 that detects the high-affinity form of LFA-1. Thus, phosphorylated Ser1140 is involved in conformational changes occurring in LFA-1 in response to several different affinity-increasing stimuli. One possibility is that the negative charge induced by phosphorylation could facilitate the separation of the integrin cytoplasmic tails, leading to a conformational change in the extracellular domain (Kim et al., 2004; Adair et al., 2005). However, a Ser-Asp mutation did not lead to a different adhesion phenotype compared with the Ser-Ala mutation, indicating that simply substituting a negatively charged amino acid for serine is not enough to mimic phosphorylation. Thus, it is more plausible that the
L phosphorylation site works through selective binding to some cytoplasmic factor.
The small GTPase Rap1 is an important mediator of various modes of LFA-1 activation in leukocytes, including activation by antibodies and divalent cations (Katagiri et al., 2000, 2002; de Bruyn et al., 2002; Sebzda et al., 2002). Importantly, Rap1 has been shown to act through the integrin chain cytoplasmic domain and to influence integrin affinity for ICAM-1 (Tohyama et al., 2003). Thus, we wanted to examine the effect of active Rap1 (Rap1V12) on the nonphosphorylatable S1140A mutant. Rap1V12 was able to induce binding of wt
Ltransfected, but not mutant-
Ltransfected, J-ß2.7 cells to coated ICAM-1. The mutated
L may be locked in a low-affinity conformation that cannot be activated from outside of the cell by activating antibodies or other activating stimuli that increase integrin affinity for its ligands. However, it can still be activated with phorbol ester and TCR stimulation, which do not (and, if so, only marginally) influence integrin affinity.
Rap1 may be working upstream of the L phosphorylation, possibly influencing the binding of some cytoplasmic factor to the
L cytoplasmic tail. RapL is a Rap1-binding molecule that can activate LFA-1 and is found in leukocytes (Katagiri et al., 2003). It will be of future interest to study the effect of RapL in this system.
It has previously been reported that the 14-3-3 isoforms ß and
from leukocyte lysates associate specifically with a Thr758-phosphorylated ß2 integrin COOH-terminal peptide (Fagerholm et al., 2002b). We discovered that the ß214-3-3 interaction is direct. Additionally, we have shown that the binding occurs in T cells that have been activated by stimuli that induce phosphorylation of the ß2 chain on Thr758, i.e., phorbol ester and TCR engagement. Mutation of Thr758 or blocking of 14-3-3 binding to the ß2 integrin by R18 peptides inhibited cell adhesion to ICAM-1. The Thr758 mutation or R18 peptide also inhibited integrin-mediated cell spreading on ICAM-1. Thus, phosphorylation of Thr758, which is induced by inside-out activating stimuli for the integrin, mediates binding to 14-3-3 proteins in cells, and this interaction seems to mediate the effect of the integrin on the cell cytoskeleton. The 14-3-3 proteins are dimers, and both monomers can independently bind to phosphorylated targets either within the same protein or in different proteins (Tzivion and Avruch, 2002; MacKintosh, 2004). It is possible that threonine phosphorylation of CD18 recruits 14-3-3 proteins to the plasma membranecytoskeleton connection and that the 14-3-3s in turn recruit other proteins to regulate downstream events.
There are increasing amounts of evidence that talin plays a profound role in activating several integrins, including LFA-1 (Kim et al., 2003; Calderwood, 2004; Smith et al., 2005). The head domain of talin binds to integrin ß chains and induces a separation of and ß chain cytoplasmic domains. Therefore, we investigated the ability of the talin head domain construct to activate the ß2-T758A mutant. It could still be activated by the talin construct in cells, as measured by increased adhesion of the transfected cells to coated ICAM-1. The talin-binding site in ß3 integrins encompasses the first NPXY motif (Tadokoro et al., 2003) and corresponds to residues 747755 in ß2, which precede Thr758. Thus, the mechanism by which Thr758 regulates adhesion is presumably not related to talin.
The cytoskeletal protein -actinin has been shown to associate with the ß2 tail at a membrane-proximal site, whereas the COOH-terminal portion of the tail (residues 748762) inhibits this interaction (Sampath et al., 1998). Substitution of Thr758 with Ala or with the phosphate-mimic Glu stimulates binding of
-actinin to ß2, suggesting that phosphorylation of ß2 on Thr758 does not regulate
-actinin binding (Sampath et al., 1998). Additionally, deletion of the
-actinin binding site in ß2 does not influence adhesion to ICAM-1. On the other hand, other proteins may bind to the TTT region and, indeed, filamin has been shown to do so (Calderwood et al., 2001). Thus, it is possible that threonine phosphorylation and 14-3-3 binding may regulate such interactions.
LFA-1 is involved in many different immunological adhesion events. It can be activated by different stimuli and is regulated both by conformational changes and by cytoskeletal attachment and clustering. It is now clear that phosphorylation of both the and ß chains plays a role in the molecular mechanisms involved in the different activation events. However, the
L and ß2 polypeptides play distinctive roles in integrin activation. The integrin
L chain is constitutively phosphorylated, and this phosphorylation site (Ser1140) seems to be required for adhesion events that involve rapid changes in the conformation and affinity of the integrin heterodimer. In contrast, the ß2-Thr758 phosphorylation that is induced after physiological triggering of T cells through the TCR works through interaction with 14-3-3 proteins. This form of adhesion is slower and involves actin reorganization and cell spreading. The contribution of the different phosphorylation events to overall adhesion may depend on the context of adhesion and provides the possibility for regulation of both fast, transient adhesive events and long-term, stable adhesion strengthening.
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Materials and methods |
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Antibodies
The mAbs R7E4 and R2E7B against the human ß2 subunit of leukocyte integrin have been described previously (Nortamo et al., 1988). The monoclonal activating antibody against CD3, OKT3, was purified from ascites fluid produced by hybridoma cells (clone CRL 8001; American Type Culture Collection). The activating mAb MEM-83 was provided by V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic). IB4 was a gift from M.A. Arnaout (Massachusetts General Hospital, Boston, MA). mAb24 (ß2) was a gift from N. Hogg (Imperial Cancer Research Fund, London, UK). The 14-3-3 antibodies H-8 and K-19, as well as the GFP antibody, were obtained from Santa Cruz Biotechnology, Inc. The HA-tagged talin F2/F3 protein was detected by antiHA-biotin antibodies (Roche) and streptavidin-HRP (GE Healthcare). To produce antiserum to phospho-L, the integrin
L chain phosphopeptide CLKPLHEKDSEpSGGGKD was conjugated to keyhole limpet hemocyanin (Harlow and Lane, 1988). The complex was injected into rabbits. The antisera were purified by affinity chromatography using the phosphorylated peptide.
cDNA constructs
The EGFP-R18 and EGFP-R18mut plasmids were gifts from T. Pawson (University of Toronto, Toronto, Canada). The 14-3-3 Saccharomyces cerevisiae isoforms BMH1 and BMH2 plasmids in DH5 were gifts from C. MacKintosh (University of Dundee, Nethergate, Dundee, Scotland, UK). The HA-tagged talin F2/F3 construct was a gift from D. Calderwood (Yale University, New Haven, CT). GFP-Rap1V12 was a gift from D. Cantrell (University of Dundee). cDNA coding for full-length human
L was subcloned into the pcDNA3 vector and human ß2 was subcloned into the
HM3 vector. Mutants were created using site-directed mutagenesis (Weiner et al., 1994), and the mutated constructs were checked by sequencing.
Cell lines and transfection
Buffy coats used for the isolation of T cells were obtained from the Finnish Red Cross Blood Transfusion Service (Valmu et al., 1999b). The cells were grown in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and antibiotics. The Jurkat cell clone E6.1 (American Type Culture Collection) was maintained in the same medium.
COS-1 cells were cultured in DME supplemented with 10% FCS, L-glutamine, and antibiotics. COS-1 cells were used for transient expression of wt and mutant ß2 integrins. COS-1 cells were cotransfected with purified - and ß-subunit cDNAs with or without EGFP-R18wt, EGFP-R18mut, or talin F2/F3 constructs using the FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche). Flow cytometric analysis was used to quantify cell surface expression of integrins in the transfected COS-1 cells.
The human T lymphoma cell line clone J-ß2.7, which lacks the LFA-1 chains and was derived from Jurkat cells by mutagenesis (Weber et al., 1997), was a gift from N. Hogg. J-ß2.7 cells (7.2 x 106 per transfection) were washed, suspended in 0.36 ml PBS, and mixed with 20 µg wt
L or S1140A-
L DNA. Electroporation was performed at 240 V and 950 µF. After 48 h of culture, the medium was supplemented with 0.8 mg/ml G418. LFA-1expressing cells were enriched using magnetic cell sorting (Miltenyi Biotec GmbH). Alternatively, cells (S1140D-
L and Rap1V12) were transfected with the Optifect system according to the manufacturer's instructions (Invitrogen). Flow cytometric analysis was used to quantify cell-surface expression of integrins in the transfected cells. For both wt
L and S1140A-
Lexpressing J-ß2.7 cells, several clones were selected that exhibited comparable levels of surface expression as detected by flow cytometry. In each experiment, we used two independent clones of both wt and
L mutant cells.
Peptide affinity chromatography
The peptides CLFKSATTTVMN and CLFKSApTTTVMN, corresponding to the ß2 integrin sequence surrounding the phosphorylated Thr758, were coupled to thiopropylSepharose according to the manufacturer's instructions. His-tagged BMH1 and BMH2 proteins were expressed in Escherichia coli DH5 and purified with Ni-NTA columns (Moorhead et al., 1999). Affinity chromatography was performed with 2 µg each of purified 14-3-3 proteins BMH1 and BMH2. After extensive washes, the bound proteins were eluted either with SDS or with 1 mM ARAApSAPA peptide or 1 mM phospho-
L peptide (CLKPLHEKDSEpSGGGKD), and the eluates were run on SDS-PAGE and stained with Coomassie blue.
14-3-3 affinity chromatography
Purified BMH1 and BMH2 or BSA were coupled to Sepharose (Moorhead et al., 1999). Jurkat cells were either left untreated or stimulated with 1.5 µM of okadaic acid, a combination of okadaic acid and the OKT3 antibody (10 µg/ml) against the TCR, or 200 nM PDBu. The cells were lysed as described previously (Valmu et al., 1999b). The lysates were mixed with the affinity matrix for 1 h and washed extensively with 500 mM NaCl. Bound proteins were eluted with SDS and analyzed by Western blotting with the blotting ß2 integrin antibody R2E7B.
Coimmunoprecipitation
Human T cells were activated with OKT3 or left untreated. Cells were lysed as described previously (Valmu et al., 1999b) and lysates were precleared with protein GSepharose. Immunoprecipitations were made with the 14-3-3 H8 antibody or control antibody (OKT3) coupled to protein GSepharose, and the immunoprecipitates were washed four times with lysis buffer. The bound proteins were eluted with SDS and analyzed by Western blotting with a 14-3-3 antibody (K-19) and ß2 integrin antibody (R2E7B).
Cell adhesion assays
Recombinant soluble human ICAM-1 or fibronectin (0.3 µg/well) was coated on flat-bottom 96-well microtiter plates by overnight incubation at 4°C. The wells were blocked with 1% dry milk for 1.5 h at 37°C. Cells were suspended in binding medium (DME for COS-1 and RPMI 1640 for J-ß2.7 cells, with 40 mM Hepes, 0.1% BSA, and 12 mM MgCl2). Cells were stimulated with either 200 nM PDBu or 10 µg/ml OKT3 or MEM-83, added to each well, and allowed to adhere for 20 (COS-1) or 30 (J-ß2.7) min at 37°C. In inhibition experiments, cells were preincubated for 15 min with 10 µg/ml of the blocking antibody R7E4. After incubation, unbound cells were removed by gentle washing. The binding was quantified by ELISA.
Immunofluorescence staining
Human T cells or J-ß2.7 cells were seeded onto poly-lysine or ICAM-1coated coverslips at 5 x 105 cells/slide in culture medium in the presence or absence of activators and incubated for 3060 min at 37°C. For transfected COS-1 spreading assays, 2 x 105 cells were seeded onto ICAM-1coated coverslips and incubated for the times indicated in the figure legends. Unbound cells were gently washed away and adherent cells were fixed for 10 min with 1% formaldehyde/PBS. Cells were labeled with FITC- or TRITC-phalloidin in 0.1% saponin/1% FBS/PBS for 2030 min, or cells were incubated with primary antibodies (mAb24, R7E4, 14-3-3 antibody K19 in PBS, or in saponin buffer) for 30 min, followed by incubation with Cy3-conjugated antimouse or FITC-conjugated antirabbit antibody. After washing with PBS, coverslips were mounted with Mowiol mounting medium and observed under a fluorescence microscope (model IX71; Olympus) and photographed with a camera (model DP70; Olympus). Images were analyzed and processed using the analySIS program (Soft Imaging System GmbH) and Adobe Photoshop.
32P radiolabeling and cell activation, immunoprecipitation, and SDS-PAGE
32P cell labeling was done as previously described (Valmu et al., 1999b). T cells were labeled overnight and COS-1 cells were labeled for 2 h at 37°C. After labeling, the cells were activated or left untreated. The T cell activation was stopped by adding ice-cold 10 mM EDTA/PBS, and COS-1 cells were detached with 5 mM EDTA/PBS. The cells were washed and lysed as described previously (Valmu et al., 1999b). The intensity of the radiolabeled bands was quantified using the Tina 2.09c software (Raytest). The intensity of the radiolabeled bands was reported as intensity of the band minus background intensity of the lane.
Cell lysates were used for immunoprecipitation with 13 µg R7E4 or IB4 for 2.5 h or overnight and coupled to protein GSepharose for 30 min at 4°C. The immunoprecipitates were washed extensively with decreasing detergent and salt concentrations. Bound proteins were eluted with 1% SDS, subjected to SDS-PAGE, and blotted onto polyvinylidene fluoride membranes (Millipore). Phosphopeptide mapping and manual radiosequencing of phosphopeptides have been described previously (Hilden et al., 2003).
Flow cytometric analysis
Cells were incubated with PBS containing 20 µg/ml of the indicated antibodies (R7E4, MEM-83, and OKT3) on ice for 30 min. The cells were then washed with PBS and further incubated with FITC-conjugated antimouse IgG and subjected to flow cytometric analysis with FACScan (Becton Dickinson). For mAb24 staining, J-ß2.7 cells or COS-cells were reacted with 10 µg/ml mAb24 in the presence of the activating antibody 10 µg/ml MEM-83 for 30 min at 37°C. Cells were instantly stained with FITC-conjugated antimouse IgG antibodies on ice for 20 min and analyzed by flow cytometry. mAb24 expression was reported as mean fluorescence intensity.
sICAM-1Fc binding assay
J-ß2.7 transfectants were incubated in 25 µl RPMI 1640, 40 mM Hepes, and 1 mM MgCl2 in the presence or absence of stimulators and 150 µg/ml ICAM-1Fc at 37°C for 30 min. After removal of the unbound ligand by washing with PBS, the cells were incubated with FITC-conjugated antihuman IgG-Fcspecific antiserum (Jackson ImmunoResearch Laboratories) on ice for 20 min. Cells were analyzed by flow cytometry.
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Acknowledgments |
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This work was supported by the Academy of Finland, The Sigrid Jusélius Foundation, the Finnish Cancer Society, and the Magnus Ehrnrooth Foundation.
Submitted: 4 April 2005
Accepted: 17 October 2005
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References |
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