Requirement of the serine at residue 329 for lipid raft recruitment of DNAM-1 (CD226)

Jun Shirakawa, Kazuko Shibuya and Akira Shibuya

Department of Immunology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences and Center for TARA, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

Correspondence to: A. Shibuya; E-mail: ashibuya{at}md.tsukuba.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Upon antigen recognition by the TCR, leukocyte function-associated antigen-1 (LFA-1) physically associates with the leukocyte adhesion molecule DNAM-1 (CD226), for which the serine phosphorylation at residue 329 (S329) of DNAM-1 plays a critical role. The TCR-mediated signal also induces the formation of the immunological synapse (IS), in which lipid raft-associated molecules, including LFA-1, DNAM-1, protein kinase C, Fyn and others, are recruited, resulting in efficient signal transduction for T cell activation. However, the molecular mechanisms of lipid raft recruitment of many associated molecules have remained unclear. Here, we demonstrate that, while both wild-type (WT) and mutant DNAM-1 at S329 were polarized at the IS, the WT, but not mutant, DNAM-1 associated with lipid rafts at the peripheral supra-molecular activation clusters. We also demonstrate that the association of DNAM-1 with lipid rafts was necessary for the tyrosine phosphorylation of DNAM-1, which is essential for LFA-1-mediated co-stimulatory signaling for naive T cell proliferation and differentiation.

Keywords: CD226, immunological synapse, LFA-1, lipid rafts


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lipid rafts were identified as detergent-insoluble membrane domains, which contain sphingolipids, sphingomyelin and cholesterol (1). These membrane microdomains play important roles in specialized pathways of membrane transport and signal transduction (2, 3). T cell activation by TCR-mediated signaling quickly induces the formation of a compartmentalized signaling cluster in lipid rafts and segregates non-raft-associated proteins from raft-associated proteins (4, 5). Lipid rafts recruit many signaling molecules, including transmembrane proteins, glycosyl-phosphatidylinositol-anchored proteins, trimeric and small GTPases, Src family tyrosine kinases, lipid signal messengers, cytosolic adaptor proteins and other signal-transducing molecules, by virtue of lipid modifications or binding to molecules that reside in lipid rafts. For example, linker for activation of T cells (LAT) Ras and Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG)/Csk-binding protein (Cbp) are palmitoylated for their targeting to lipid rafts (6, 7). Miristoylation and palmitoylation of alpha subunits of G protein, Src family tyrosine kinases and Nos are necessary for partitioning into lipid rafts. However, molecular mechanisms of lipid raft recruitment of many associated molecules have remained unclear.

Lipid rafts are thought to play an important role in forming the structure of the immunological synapse (IS), which is a specialized signaling domain characterized by complex molecular clustering and segregation at the contact site between a T lymphocyte and an antigen-presenting cell (APC) (8). Upon antigen recognition by TCR, leukocyte function-associated antigen-1 (LFA-1) is recruited to lipid rafts and is involved in the formation of peripheral supra-molecular activation clusters (p-SMAC) that surround a central cluster with TCR–MHC peptide complex, called central SMAC (c-SMAC), at the IS (9). Interaction of LFA-1 on T cells with inter-cellular adhesion molecules (ICAM) on APC at the IS not only mediates intercellular binding but also delivers co-stimulatory signals in T lymphocytes.

The leukocyte adhesion molecule DNAM-1 is constitutively expressed on the majority of T lymphocytes, NK cells, monocytes and platelets (10, 11). Stimulation of peripheral T cells with anti-CD3 induces the physical association of LFA-1 with DNAM-1 at the IS, for which the serine phosphorylation of DNAM-1 at residue 329 is responsible (12, 13). Once LFA-1 and DNAM-1 associate, cross-linking LFA-1 induces tyrosine phosphorylation of DNAM-1 at residue 322, for which the Fyn protein tyrosine kinase (PTK) is responsible (12), and mediates co-stimulatory signals for triggering naive T cell differentiation into Th1 phenotype and proliferation (11). We have recently reported that, upon stimulation of CD4+ T cells with beads pre-coated with anti-CD3 plus either anti-CD28 or anti-LFA-1 mAbs, DNAM-1 as well as LFA-1 and Fyn are recruited in lipid rafts and polarized at the IS (13). However, the molecular mechanism by which DNAM-1 is recruited into lipid rafts and involved in LFA-1-mediated signal transduction remains undetermined.

In the present study, we investigated the molecular mechanism of lipid raft recruitment of DNAM-1. We demonstrate that the serine phosphorylation of DNAM-1 at residue 329 plays a critical role for lipid raft recruitment and LFA-1-mediated signaling.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells, reagents and antibodies
Raji cells were obtained from RIKEN BRC (Tsukuba, Japan). CD4+ T cells were separated from cord blood by positive selection using magnetic cell sorting (MACS). The purity of CD4+ T cells was >95%, as analyzed by flow cytometry. For Jurkat T cell stimulation, cells were incubated with 10 µg ml–1 anti-CD3{varepsilon} and anti-CD28 on ice for 20 min, followed by 15 µg ml–1 anti-mouse IgG rabbit polyclonal antibody at 37°C for 5 min. Phorbol 12-myristate 13-acetate (PMA) and anti-Flag mAb were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bisindolylmaleimide I (BIM) was purchased from Calbiochem (San Diego, CA, USA). Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Anti-DNAM-1 mAb (DX11) was provided by Joe Phillips (DNAX Research Institute, Palo Alto, CA, USA). The other mAbs used in this study were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD3, anti-CD11a and anti-DNAM-1 mAbs were labeled with Alexa594 by Monoclonal Antibody Labeling Kit (Molecular Probes, Eugene, OR, USA).

Establishment of Jurkat transfectants with Flag-tagged wild-type and mutant DNAM-1
Flag-tagged wild-type (WT) and mutant (Y-F322 and S-F329) DNAM-1 cDNAs were described previously (13, 14). The cDNAs were sub-cloned into the pGC-sap retroviral vector (15). Jurkat cells or cord blood CD4+ T cells stably expressing Flag-tagged WT and mutant DNAM-1 were established, as described (12). Expressions of WT and mutant DNAM-1 of Jurkat transfectants were comparable with each other, as analyzed by flow cytometry using anti-Flag mAb.

Lipid rafts isolation and biochemistry
Lipid rafts were isolated by discontinuous sucrose density gradient ultracentrifugation, by a modified protocol as described (5). In brief, 2 x 107 Jurkat cells were lysed in 1 ml of ice-cold 1% Brij-35 or 0.3% Triton X-100 in TN buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA) with protease inhibitors (1 mM phenylmethylsulfonylfluoride, 15.3 TIU aprotinin) for 30 min, gently mixed with an equal volume of 85% in TN buffer and placed in the bottom of a SW41i centrifuge tube (Becton Dickinson Mountein View, CA, USA). The sample was then overlaid with 5 ml 35% sucrose and 4 ml 5% sucrose in TN buffer and spun for 17–19 h at 200 000 x g at 4°C. Each 1 ml gradient fraction was sequentially collected from the top of the gradient. Sucrose density fractions of cell lysates were immunoprecipitated with anti-Flag and proteins were separated by SDSP. Immunoblotting analyses were performed, as described (12, 14).

Immunofluorescence
For studies of lipid raft capping, Jurkat cells or Jurkat transfectants were stimulated or not with PMA (50 ng ml–1) at 37°C for 2 h, stained with FITC-conjugated cholera toxin subunit B (FITC-CTx) (Sigma–Aldrich) at 4°C for 20 min and then cross-linked with anti-CTx antibody (Calbiochem) at 4°C for 30 min. Cells were then incubated at 37°C for 20 min, transferred onto poly-L-lysine pre-coated cover slips and incubated at 37°C for 5 min. For T cell–APC conjugate formation, Raji cells were pulsed with 200 ng ml–1 superantigen staphylococcal enterotoxin E (SEE) (Toxin Technologies, Sarasota, FL, USA) at 37°C for 30 min and then mixed with an equal number of Jurkat transfectants that had been stained with FITC-CTx. Then, cells were centrifuged at low speed and incubated for 15 min at 37°C. Cell conjugates were gently resuspended, plated onto poly-L-lysine pre-coated cover slips and incubated at 37°C for 5 min.

Cells on cover slips, after pre-treatment as described above, were fixed with 2% formaldehyde and stained with biotin-conjugated anti-FLAG or anti-DNAM-1 mAbs, followed by Streptavidin-Alexa647 (Molecular Probes). Cover slips were mounted with Slow Fade (Molecular Probes) and cells were analyzed by using a Leica TCS SP2 confocal laser scanning microscope.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Co-localization of DNAM-1 with lipid rafts
We have recently reported that, upon stimulation of primary CD4+ T cells with anti-CD3 plus either anti-CD28 or anti-LFA-1 mAbs-coated beads, DNAM-1 as well as LFA-1 and Fyn were polarized at the IS, in which lipid rafts were also aggregated (13), suggesting that DNAM-1 is recruited into lipid rafts. To confirm the DNAM-1 recruitment into lipid raft compartment, ganglioside type 1 (GM-1) on Jurkat cells was cross-linked with CTx and anti-CTx mAb and then Jurkat cells were stained with anti-DNAM-1 mAb. As demonstrated in Fig. 1, cross-linking GM-1 resulted in capping and co-localization of GM-1 and DNAM-1. Jurkat cells were also stimulated with beads pre-coated with anti-CTx or anti-CD3 plus anti-CD28 mAbs and stained with anti-CTx and either anti-DNAM-1 or anti-LFA-1 mAbs, demonstrating the aggregation of GM-1, DNAM-1 and LFA-1 at the contact site with beads (Fig. 1). More than 90% of DNAM-1 was accumulated at the contact site, as determined by using NIH image of >20 cell-bead conjugates. These results suggest that DNAM-1 as well as LFA-1 are recruited into the lipid raft compartment in Jurkat cell.



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Fig. 1. Co-localization of DNAM-1 with lipid rafts. Jurkat cells were stained with FITC-CTx, followed by cross-linking with anti-CTx antibody (upper panel) or by stimulation with beads pre-coated with anti-CTx (the upper second panel) or anti-CD3 plus anti-CD28 mAbs (the third and lower panel). Jurkat cells were then fixed with formaldehyde and stained with Alexa594-conjugated anti-DNAM-1 or LFA-1 (CD11a) mAbs. Data are representative of >100 cells observed in each experiment.

 
The serine at residue 329 (S329) is required for lipid raft recruitment of DNAM-1
Because S329 of DNAM-1 is required for physical association of DNAM-1 with LFA-1 on T cells after stimulation with anti-CD3 mAb (12), we examined whether the serine mutation at the residue affects lipid raft recruitment of DNAM-1. We established Jurkat transfectants expressing WT or mutant (S-F329) DNAM-1 tagged with Flag at the N-terminus. The expressions of cell surface DNAM-1 of these transfectants were comparable, as analyzed by flow cytometry using anti-Flag mAb (Fig. 2A). To demonstrate the association of DNAM-1 with lipid rafts, we first used 0.3% Triton X-100 as a lysis buffer for the transfectant expressing WT DNAM-1. The lysates were fractionated by discontinuous sucrose density gradient ultracentrifugation. Immunoblot analyses of each fraction demonstrated that WT DNAM-1 was detected only in Triton X-100-soluble (fractions 10–11), but not insoluble (fractions 4–5), fractions (Fig. 2B). Because LFA-1 on primary T cells could be detected in Brij-35-insoluble, but not in Triton X-100-insoluble, lipid rafts (16), we next used 1% Brij-35 for lysis of the transfectant. As demonstrated in Fig. 2(B), WT DNAM-1 was detected in both Brij-35-insoluble and -soluble fractions. The recruitment of WT DNAM-1 in the Brij-35-insoluble fractions in the Jurkat transfectant did not change after stimulation of the transfectant with anti-CD3 and anti-CD28. We also detected CTx-binding GM-1 and lipid raft-associated molecules such as PTKs Fyn and Lck, but not CD45, in the Brij-35-insoluble fractions (Fig. 2C). In contrast, although immunoblotting analysis by using anti-DNAM-1 mAb showed the recruitment of DNAM-1 including endogenous WT DNAM-1 in both Brij-35-insoluble and -soluble fractions, S-F329 mutant DNAM-1 was detected only in Brij-35-soluble fractions by using anti-Flag mAb even after stimulation of the transfectant with anti-CD3 and anti-CD28 mAbs (Fig. 2B). We observed similar results when we used cord blood CD4+ T cells, instead of Jurkat cells, transfected with WT or S-F329 mutant DNAM-1 (Fig. 2B). Taken together, these results indicate that WT, but not S-F329 mutant, DNAM-1 associates with 1% Brij-35-insoluble lipid rafts.



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Fig. 2. S-F329 mutant DNAM-1 does not associate with lipid rafts. A. Jurkat cells (dotted line) or Jurkat transfectants stably expressing Flag-tagged mutant DNAM-1 indicated were stained with anti-Flag mAb, followed by PE-conjugated secondary antibody. B, C. Jurkat cells or cord blood CD4+ T cells transfected with Flag-tagged WT or S-F329 mutant DNAM-1 were stimulated or not with anti-CD3 plus anti-CD28 mAbs, or pre-incubated in the presence of BIM for 3 h, and lysed with 0.3% Triton X-100 or 1% Brij-35 at 4°C and fractionated on a sucrose density gradient, as described in Methods. Each fraction was immunoblotted with CTx or mAbs indicated.

 
We previously reported that S329 was constitutively phosphorylated in Jurkat T cells (12). While S-F329 mutant DNAM-1 was never recruited in lipid raft fractions in Jurkat transfectant, endogenous WT DNAM-1 of the transfectant was recruited (Fig. 2B). Moreover, a protein kinase C (PKC) inhibitor completely abrogated the lipid raft recruitment of DNAM-1 in Jurkat transfectant (Fig. 2B). These results suggest that S329 phosphorylation of DNAM-1 may play an important role for the association of DNAM-1 with lipid rafts.

S329 is not required for DNAM-1 translocation to the IS
To examine whether S329 is also involved in DNAM-1 polarization at the IS, Jurkat transfectants expressing WT or S-F329 mutant DNAM-1 were co-cultured with SEE-pre-pulsed Raji cells that were used as APC. As demonstrated in Fig. 3, both WT and S-F329 mutant DNAM-1 and GM-1 were aggregated at the contact site with APC, indicating that the S329 is not required for DNAM-1 polarization at the IS. It was of note, however, that S-F329 mutant DNAM-1 did not co-localize with GM-1, but rather surrounded the GM-1 compartment at the IS, demonstrating again that S329 is required for DNAM-1 recruitment into lipid rafts compartment.



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Fig. 3. Lipid raft-independent translocation of DNAM-1 to the ISA. Jurkat transfectants expressing Flag-tagged WT, S-F329 or Y-F322 mutant DNAM-1 were stained or not with either FITC-conjugated CTx. Cells were then co-cultured with SEE-pre-pulsed Raji cells used as APC for 15 min and these cell mixtures were stained with biotin-labeled anti-Flag mAb, followed by Alexa647-conjugated streptavidin. Otherwise, Jurkat transfectants were co-cultured with SEE-pre-pulsed Raji cells and then stained with biotin-labeled anti-Flag mAb and either FITC-conjugated anti-LFA-1 (CD11a) or anti-CD3, followed by Alexa647-conjugated streptavidin. It is of note that S-F329 mutant DNAM-1 (red arrow head) segregated from lipid rafts or LFA-1 (green arrow head). Three-dimensionally reconstructed T-APC contact sites are also shown in B. Each datum is representative of >100 T-APC conjugates observed in each experiment.

 
To examine where DNAM-1 is distributed in the IS, DNAM-1 localization was compared with those of CD3 and LFA-1, which are involved in the formation of c-SMAC and p-SMAC, respectively (9). When Jurkat cells expressing WT DNAM-1 were co-cultured with SEE-pre-pulsed Raji cells, DNAM-1 co-localized with GM-1 and LFA-1 at the IS (Fig. 3A), indicating that DNAM-1 was distributed at p-SMAC. However, although S-F329 mutant DNAM-1 was aggregated at the IS, it did not co-localize with GM-1 and LFA-1, but rather surrounded the LFA-1 compartment at the IS (Fig. 3A). These observations were further confirmed by the three-dimensional analysis of the IS, showing that while WT DNAM-1 was overlapped with LFA-1, S-F329 mutant DNAM-1 was localized outside LFA-1 at the contact site between the Jurkat transfectant and APC (Fig. 3B).

Phosphorylation of the tyrosine322 of DNAM-1 requires association with lipid rafts
Our prior studies demonstrated that cross-linking TCR and LFA-1 with antibodies induces activation of Fyn that is then recruited to DNAM-1, resulting in the tyrosine phosphorylation of DNAM-1 at residue 322 (12). Introduction of the tyrosine mutant (Y-F322) CD226 cDNA into naive CD4+ T cells by lentiviral vector inhibited LFA-1-mediated co-stimulatory signals for their differentiation and proliferation (13), indicating that the tyrosine phosphorylation of CD226 plays a critical role for LFA-1-mediated signaling. Because Fyn was detected in both raft and non-raft fractions in the Jurkat transfectant (Fig. 2C), we examined whether DNAM-1 is tyrosine phosphorylated in either raft or non-raft fractions or in both. As demonstrated in Fig. 4, although considerable amount of WT DNAM-1 was detected in both fractions, the tyrosine phosphorylation of DNAM-1 was detected exclusively in raft, but not non-raft, fractions. These results are in accordance with a recent report that the Fyn kinase activity that is responsible for Y322 phosphorylation of DNAM-1 was highly enriched in lipid raft fractions (12, 17). These results suggest that DNAM-1 is tyrosine phosphorylated exclusively in raft fractions. It is of note that, although Y-F322 DNAM-1 was not tyrosine phosphorylated (Fig. 4), this mutant DNAM-1 was also detected in lipid raft compartment at the IS (Fig. 3), indicating that the tyrosine phosphorylations of DNAM-1 is not necessary for lipid rafts recruitment of DNAM-1. In addition, the serine phosphorylations of both WT and Y-F322 DNAM-1 were detected in both raft and non-raft fractions (Fig. 4), suggesting that S329 phosphorylation of DNAM-1 is not sufficient for lipid raft recruitment of DNAM-1.



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Fig. 4. Tyrosine phosphorylation of DNAM-1 occurs in lipid rafts. Jurkat transfectants expressing Flag-tagged WT, S-F329 or Y-F322 mutant DNAM-1 were lysed with 1% Brij-35 at 4°C and fractionated on a sucrose density gradient, as described in Methods. Lipid raft fractions (fractions 4–5) and non-lipid raft fractions (fractions 10–11) were immunoprecipitated with anti-Flag mAb and immunoblotted with anti-Flag, anti-phosphotyrosine or anti-phosphoserine antibodies. Data are representative of several independent experiments.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we have shown that DNAM-1 recruitment into the lipid raft compartment requires S329, which is likely phosphorylated by PKC. It was reported that PKC{theta} and PKC{gamma}-1 translocate from the cytosol to lipid rafts during TCR signaling (1820), suggesting that these kinases may be candidates responsible for the serine phosphorylation of DNAM-1. We observed that the serine phosphorylation of DNAM-1 was not inhibited in the presence of Rottlerin, a specific inhibitor for PKC{delta} and PKC{theta} (data not shown). However, we also observed that lipid raft recruitment of DNAM-1 was completely abrogated in the presence of BIM, an inhibitor with broad specificities for PKC subtypes (Fig. 2B). It was also reported that B cell receptor-dependent recruitment of I kappa B kinase (IKK) to the lipid raft compartment requires a PKC (PKCß) in B cells (21). Although how the serine phosphorylation leads to the association of DNAM-1 with lipid rafts remains undetermined, identification of a PKC subtype responsible for lipid rafts recruitment of DNAM-1 may contribute to further elucidation of its molecular mechanism.

A recent report demonstrated that, although antigen-induced translocation of lipid rafts to the IS was severely impaired in DOCK2-deficient T cells, translocation of LFA-1 and PKC{theta} to the IS was intact (22), indicating that lipid rafts are not always ‘rafts’ for their translocation to the IS. In the present study, we have shown that TCR-mediated signaling induced translocation of the S-F329 mutant, as well as WT, DNAM-1 to the IS; however, the mutant DNAM-1 was segregated from GM-1-rich lipid rafts. These results indicate that DNAM-1 also does not require GM-1-rich lipid rafts for translocation to the IS. It is of interest that LFA-1 can be detected in Brij-35-insoluble, but not Triton X-100-insoluble, fractions in T cells (16). In the present study, we also observed a similar result, i.e. DNAM-1 was detected in Brij-35-insoluble, but not Triton X-100-insoluble, fractions. Because Brij-35-insoluble fractions contain a higher level of cholesterol and lower level of GM-1 than Triton X-100-insoluble fractions, TCR-mediated signaling may induce translocations of LFA-1 and DNAM-1 to the IS by using cholesterol-rich membrane microdomains different from the GM-1-rich compartment. Nonetheless, association of DNAM-1 with lipid rafts is important for both DNAM-1 and LFA-1 because tyrosine phosphorylated DNAM-1, which is essential for LFA-1-mediated co-stimulatory signaling in naive T cells (13), was exclusively present in this fraction. A similar model has recently been reported that integrity of lipid rafts was essential for phosphorylation and activation of NK cell-activating receptor 2B4 (CD244) (23).

Burns and colleagues have very recently identified the actin-binding protein 4.1G and the membrane-associated guanylate kinase homologue, human discs large (hDlg), as associating molecules with DNAM-1 (24). They also showed that T cell stimulation with phorbol ester induced DNAM-1 and 4.1G to associate tightly with Rap1 and the cytoskeleton, respectively (24). Kinashi and colleagues previously described that T cell stimulation also induces the association of Rap1 with Rap1 ligand (RAPL), followed by binding of these associating molecules with LFA-1 (25). These reports together with our prior studies suggest that the TCR-mediated signal induces a large complex formation, consisting of DNAM-1, Fyn, 4.1G, hDlg, Rap1, RAPL, LFA-1 and the actin cytoskeleton. To clarify the regulatory mechanisms of DNAM-1 recruitment into lipid rafts, it should be required to further elucidate the signaling pathways of this large complex.


    Acknowledgements
 
We thank Lewis Lanier for critical reading of the manuscript. We also thank Tomie Kameyama, Satoshi Yamazaki and Kensuke Yamazaki for technical assistance, Yurika Soeda for secretarial assistance and the members of our laboratory for helpful discussion. This research was supported in part by the grants provided by the Ministry of Education, Science and Culture of Japan (to A.S. and K.S.), Special Coordination Funds of the Science and Technology Agency of the Japanese Government (to A.S.), the Uehara Memorial Foundation (to A.S.) and the Yasuda Memorial Foundation (to A.S.).


    Abbreviations
 
APC   antigen-presenting cell
BIM   bisindolylmaleimide I
c-SMAC   central supra-molecular activation clusters
FITC-CTx   FITC-conjugated cholera toxin subunit B
GM-1   ganglioside type 1
hDlg   human discs large
IS   immunological synapse
LFA   leukocyte function-associated antigen
PKC   protein kinase C
PMA   phorbol 12-myristate 13-acetate
p-SMAC   peripheral supra-molecular activation clusters
PTK   protein tyrosine kinase
RAPL   Rap1 ligand
SEE   staphylococcal enterotoxin E
WT   wild type

    Notes
 
Transmitting editor: T. Saito

Received 8 September 2004, accepted 22 November 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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