Proc. Nail. Acad. Sci. USA Vol. 88, pp. 9132-9135, October 1991
Immunology
Fyn tyrosine kinase associated with FceRII/CD23: Possible multiple roles in lymphocyte activation (YT cells/interleukin 2 receptor/pSS)
KATSUJI SUGIE*t, TOSHIAKI
KAWAKAMI0§, YASUHIRO MAEDA*, TAKUMI KAWABE*, ATSUSHI UCHIDAt,
AND JUNJI YODOI*¶ *Department of Biological Responses, Institute for Virus Research, tDepartment of Late Effect Studies, Radiation Biology Center, and tDepartment of Medical Chemistry, Kyoto University Medical School, Sakyo-ku, Kyoto 606-01, Japan
Communicated by Kimishige Ishizaka, July 17, 1991
Expression of low-affinity Fc receptor for IgE ABSTRACT (FceRll), which is identical to the lymphocyte differentiation antigen CD23, is associated with activation of lymphoid cells. The mechanism of signal transduction through FceRll/CD23 was dissected by transfection of cDNA coding for FccRII to the YT human natural killer-like cell line, activation of which was easily detected by the induction of interleukin 2 receptor/p55(Tac). Cross-linking of FceRII/CD23 with H107 antiFceRll monoclonal antibody markedly enhanced interleukin 2 receptor/p55 expression on the YT cells transfected with FceRH cDNA (YTSER cells). Similar induction of interleukin 2 receptor/p55 by the cross-linking of FcERll was observed on an Epstein-Barr virus-transformed B-cell line, 3B6, and fresh leukemic cells isolated from a patient with B-cell chronic lymphoblastic leukemia. Thus FcERll/CD23 provides activation signals not only in YTSER cells but also in activated B cells. A possible involvement of protein-tyrosine kinase in the FcERII-mediated signal transduction was studied. FcERII was physically associated with a src family tyrosine kinase p59" and not with p56'k, which was also found in YT cells. Recently it was reported that p59r-v was associated with T-cell antigen receptor. Our results collectively suggest the multiple functions of p59fyI that may be implicated in FcERll-mediated activation signal in YT cells.
to indicate direct interactions of FceRI1 molecule with GTPbinding proteins. FceRII has no protein-tyrosine kinase (PTK) domain, whereas other receptors such as plateletderived growth factor receptor and epidermal growth factor receptor were reported to phosphorylate the phospholipase C-y chain (16). In an attempt to define the signal transduction mechanism through FceRII, we transfected cDNA encoding FceRIl (17) to YT cells and established the YTSER cell line constitutively expressing the molecule on the cell surface. Interleukin 2 receptor (IL-2R)/p55(Tac) is induced on YT cells by various stimuli, including cytokines (18, 19) and pharmacological agents (20-22). Cross-linking of FceRII on YTSER cells with H107 anti-FceRII mAb resulted in enhanced expression of IL-2R/p55(Tac). Since PTKs are deeply involved in the growth regulation of various cell types, including lymphoid cells (23), we next tested possible involvement of PTK in FceRII-mediated cell activation. The present experiments show that FceRII on YTSER cells is physically associated with a src family PTK p59fyn, which was recently reported to associate with T-cell antigen receptor (TCR) (24). Possible multiple functions of PTK associated with lymphocyte receptors are discussed.
The low-affinity Fc receptor for IgE (FceRII) is involved not only in the regulation of IgE production (1, 2) but also in the activation or transformation of lymphoid cells, particularly of B-cell lineage. Indeed, FceRII is identical to CD23 (3, 4), which is strongly expressed on Epstein-Barr virus (EBV)transformed B lymphoblast (5, 6). Accumulating evidence suggests that cross-linking of FceRII/CD23 results in signal transduction, which regulates B-cell growth (7, 8). However, biological roles of FcERII/CD23 on non-B cells (9-12) are poorly understood. We have recently found that stimulation of the YT human natural killer (NK)-like cell line (13) with recombinant interleukin 1,B induced FcERII/CD23 (K.S. and Y.M., unpublished observations), suggesting a possible role of the molecule in the activation of NK cells. Interestingly, a cell-surface molecule related to NK function has been reported to show a significant homology with the C-type animal lectins such as FceRII/CD23 (14). These findings collectively indicate the important roles of FcERII/CD23 in various kinds of lymphocyte activation, whereas the molecular mechanism involved in the signal transduction through FceRII remains elusive. It was recently reported that antiCD23 monoclonal antibodies (mAbs) induced a rise in intracellular [Ca2+] and inositol phospholipid hydrolysis in human activated B cells (15). However, no evidence has been shown
MATERIALS AND METHODS Cells and Antibodies. 3B6 (25) is an EBV-transformed B-cell line kindly provided by H. Wakasugi (Institute Gustave Roussy, Villejuif, France). HUT-102 is a human T-lymphotropic virus, type I-positive T-cell line. Fresh leukemic cells of B-cell chronic lymphoblastic leukemia (BCLL) were isolated from peripheral blood of a patient by Ficoll-Paque (Pharmacia). All cells were maintained in RPMI 1640 medium (GIBCO) containing 10% heat-inactivated fetal calf serum (Whittaker Bioproducts) and antibiotics (100 units of penicillin per ml and 100 gg of streptomycin per ml). H107 anti-FceRII mouse IgG2b mAb (26) was purified from ascitic fluid by using protein A-Sepharose affinity chromatography. Antisera against p56lck (anti-LCK) and N-terminal residues 29-43 of p59fY (anti-FYN N) were raised in rabbits as described (27). Anti-Tac mAb and rabbit anti-phosphotyrosine antiserum were generously provided by T. Uchiyama (Kyoto University) and D. P. Bottaro (National Cancer Institute), respectively. PY-20 anti-phosphotyrosine mAb was purchased from ICN. Abbreviations: mAb, monoclonal antibody; FceRII, low-affinity Fc receptor for IgE; NK, natural killer; IL-2R, interleukin 2 receptor; PTK, protein-tyrosine kinase; TCR, T-cell antigen receptor; EBV, Epstein-Barr virus; B-CLL, B-cell chronic lymphoblastic leukemia. §Present address: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037. ITo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9132
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Proc. Nati. Acad. Sci. USA 88 (1991)
Transfection of FcERll/CD23 cDNA. The EcoRI-digested fragment of pKCReSER, including simian virus 40 early promoter/enhancer and FceRII cDNA (17), was inserted into the EcoRI site of pSV2neo (pSV2neoSER). Subclones of YT cells stably transformed with pSV2neoSER (YTSER cells) or pSV2neo (YTNEO-A1 and YTNEO-B1) were established as follows. YT cells (5 x 106) were transfected with 10 Ag of pSV2neoSER or pSV2neo at 2000 V and 8-.uF pulse in cuvettes (Sarstedt) by using an electric cell borer (Richo Chemical Research, Kyoto). After preculture in RPMI 1640 medium containing 10%o fetal calf serum for 48 hr. the transformed cells were maintained in complete culture medium with 0.4 mg of G418 per ml (Sigma) for 4 wk. After selection, the cells transfected with pSV2neoSER (YTSER) were positive for FceRII, as determined by flow cytometry, whereas those transfected with pSV2neo (YTNEO-A1 and YTNEO-B1) were negative (Fig. 1). IL-2R/p55(Tac) Induction. Cells were preincubated on ice with 20 Ag of H107 mAb or control mouse IgG2b (Cappel Laboratories) per ml for 30 min, washed, and cultured with or without 10 ug of goat anti-mouse IgG antibodies per ml (Cappel) for 24 hr. IL-2R/p55(Tac) expression was examined by flow cytometry and Northern blot analysis as described elsewhere (10). Immunoblotting Using Anti-Phosphotyrosine Antiserum. Cells (1.5 x 107) were lysed in 1% Nonidet P-40, 20 mM Tris-HCI (pH 7.2), 150 mM NaCl, 0.1% NaN3, 1 mM phenylmethylsulfonyl fluoride (Calbiochem), 3.3 ug of aprotinin per ml (Sigma), and 0.2 mM sodium orthovanadate (Sigma). The lysates were incubated with H107 mAb or control mouse IgG2b, and immunoprecipitates were recovered with the aid of Pansorbin (Calbiochem). Precipitated proteins were separated on a 10%6 NaDodSO4/polyacrylamide gel and transferred to a poly(vinylidene difluoride) sheet, Immobilon (Millipore). The blots were incubated with rabbit anti-phosphotyrosine antiserum, washed, and treated with '251-labeled protein A ('25Iprotein A; ICN) followed by autoradiography. In Vitro Kinase Assays. In vitro kinase assays were performed by a modification of the method described previously 100
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FcERIIa cDNA was inserted into the EcoRI site of pSV2neo vector and transfected by electroporation. Expression of FceRIIa cDNA was regulated by simian virus 40 early promoter/enhancer systems. After selection with G418, pSV2neoSER transfectants (YTSER cells) expressed FcERII/CD23, as determined by flow cytometry, whereas pSV2neo transfectants (YTNEO-A1 and YTNEO-Bl) did not. A representative result is shown from three experiments with similar results.
9133
(28). In brief, phosphotyrosine-containing proteins were affinity-purified from lysates of unlabeled cells by using PY20 mAb and subjected to immunoprecipitation by H107 mAb or control IgG2b. Immunoprecipitates were incubated at 250C for 10 min in 0.025 ml of 20 mM Tris HCl (pH 7.2)/5 mM MnCl2 containing 10 1Ci of fy-32PJATP (3000 Ci/mmol; 1 Ci = 37 GBq) and analyzed by 10% NaDodSO4/polyacrylamide gel electrophoresis followed by autoradiography. After in vitro kinase assays, 32P-labeled proteins were subjected to phosphoamino acid analysis as described elsewhere (29). Peptide Mapping. The gel slices after in vitro kinase assays were overlaid with 0.5 ,ug of V8 protease per ml (ICN) and electrophoresed on a 12.5% NaDodSO4/polyacrylamide gel. RESULTS To determine whether FcERII expressed on YTSER cells could transmit activation signals, we measured IL-2R/ p55(Tac) expressed on YTSER cells after stimulation with H107 anti-FcERII mAb. When YTSER cells were treated with H107 mAb and then cross-linked by polyclonal goat anti-mouse IgG antibodies, increased expression of IL-2R/ p55 was detected by flow cytometry (Fig. 2A). In contrast, the treatment with H107 mAb alone or H107 mAb followed by the second antibody did not enhance IL-2R/pS5 expression on parental YT cells. Northern blot analysis also showed that the level of IL-2R/pS5 mRNA in YTSER was upregulated by triggering the cells with H107 mAb and the second antibody (Fig. 2B). These results on the YTSER transfectant raised the possibility that the endogenous FceRII/CD23 on lymphoid cells may also transmit a signal for the induction of IL-2R/p55. As shown in Fig. 2A, cross-linking of the endogenous FceRII expressed on the 3B6 EBV-transformed B-cell line resulted in IL-2R/p55 induction. The cross-linking also induced IL2R/p55 significantly on fresh leukemic cells from a patient with B-CLL. These data collectively indicate that endogenous and transfected FceRII/CD23 could transmit the activation signal into the cells. Since protein-tyrosine phosphorylation has been implicated to play a crucial role in the signal transduction through various cell-surface receptors, we next examined whether tyrosine phosphorylation is involved in FceRII-mediated cell activation. To test whether proteins phosphorylated at tyrosine residues are associated with FceRII in YTSER cells, an immunoblot analysis was performed by the use of polyclonal anti-phosphotyrosine antibody. Several tyrosine-phosphorylated proteins were coimmunoprecipitated by H107 mAb from YTSER cells (Fig. 3A). The most prominent phosphoprotein had a relative molecular mass of 59 kDa (p59). No such coimmunoprecipitation was seen with control clones (YTNEO-A1, YTNEO-B1) or parental YT cells. The results indicate that p59 phosphorylated at tyrosine residues was associated with FcERII on YTSER cells. We next performed in vitro kinase assays to ascertain that the CD23/H107 immune complexes have protein kinase activity. Addition of [y-32P]ATP to the H107-precipitated immune complex from YTSER resulted in phosphorylation of a 59-kDa protein (Fig. 3B, lane 1). In contrast, no phosphorylated product was detected in the parental YT cells (lane 2) or in immunoprecipitates from YTSER cell lysates with control IgG2b (lane 3). A phosphoamino acid analysis of the 59-kDa protein phosphorylated in in vitro kinase assays showed that it was phosphorylated exclusively on tyrosine residues (Fig. 3C). It is thus evident that FcERII of YTSER cells is physically associated with a PTK. A similar association between FceRII and PTK was observed in several FcsRII-positive B-lymphoid cell lines (data not shown). The size of the p59 molecule that is associated with FcERII on YTSER cells falls in the range of the src family PTKs.
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Immunology: Sugie et al.
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FIG. 2. IL-2R/p55 induction through FceRII. (A) Cells were preincubated on ice with 20 fxg of H107 anti-FcERII mAb (solid lines) or control IgG2b (dashed lines) per ml, washed, and cultured with or without 10 pyg of goat anti-mouse IgG per ml for 24 hr. IL-2R/ p55(Tac) expression was estimated by flow cytometry on a linear scale. Gain was adjusted so that Tac-positive populations before stimulation were around 10%o. Only a representative result is shown among three experiments with similar results. (B) YTSER cells were stimulated (as described in A) with medium alone (lane 1), control IgG2b plus anti-mouse IgG (lane 2), H107 (lane 3), or H107 plus anti-mouse IgG (lane 4). IUT102 (lane 5) is a human T-lymphotropic virus, type I-positive T-cell line. Total cellular RNA was analyzed by Northern blotting using 32P-labeled IL-2R/p55 cDNA probe. Homogeneity of the loaded amounts of RNA was verified by hybridization of the same blot with a pseudo-actin probe. kb, Kilobases.
Using the immune complex kinase assays, we have detected p561ck and p59f" in YT cells (K.S. and T.K., unpublished observations). To determine whether the p59 is identical to p56Ick or p59fYn, we performed V8 protease mapping. Immune complexes prepared from YTSER using antibodies directed against FceRII (H107), p56lck, or p59fyf were subjected to in vitro kinase assays and analyzed on a NaDodSO4/polyacrylamide gel. After autoradiography, the bands of phosphoproteins of56-59 kDa were excised from the gel and exposed to V8 protease. Fig. 4A demonstrated that the pattern of bands derived from partial digestion ofthe p59 was almost identical to that of p59fyt but distinct from that of p561ck. To further confirm that the FceRII-associated p59 is identical to p59fyn, YTSER cell lysates were preabsorbed with anti-p59'yn or anti-p56ck plus fixed staphylococcal Cowan I (Pansorbin), and FceRII was immunoprecipitated with H107 mAb. The in vitro kinase assays demonstrated that the p59 was completely depleted by preabsorption with anti-p599Z but not with anti-p56lck (Fig. 4B).
FIG. 3. Association of PTK with FceRII on YTSER. (A) Cells (1.5 x 107) of YTSER (lanes 1 and 2), YTNEO-A1 (lanes 3 and 4), YTNEO-B1 (lanes 5 and 6), and parental YT (lanes 7 and 8) were solubilized in 1% Nonidet P.40, 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 3.3 mg of aprotinin per ml, and 0.2 mM sodium orthovanadate. Proteins immunoprecipitated with H107 mAb (lanes 1, 3, 5, and 7) or control IgG2b (lanes 2, 4, 6,
and 8) were washed in the lysis buffer, separated on 10%1 NaDodSO4/ polyacrylamide gels, transferred, and probed with a rabbit antiphosphotyrosine antibody (provided by D. P. Bottaro)followed by detection with 1251-protein A. The specificity of the anti-phosphotyrosine antibody was confirmed by including p-nitrophenyl phosphate in the incubation with the antibody. No discernible bands were detected (data not shown). A typical autoradiograph is shown (out of three similar experiments). (B) Phosphotyrosine-containing proteins were affinity-purified from YTSER (lanes 1 and 3) or parental YT (lane 2) with anti-phosphotyrosine mAb PY20 and incubated with H107 mAb (lanes 1 and 2) or control IgG2b (lane 3). Immunoprecipitates were subjected to in vitro kinase assays, in which samples were incubated in 20 mM. Tris-HCl (pH 7.2), 5 mM MnCl2, and 10 ,uCi of [y-t32P]ATP and separated on 10%o NaDodSO4/ polyacrylamide gels. (C) After in vitro kinase assays, 32P-labeled p59 from YTSER was subjected to phosphoamino acid analysis. Positions of phosphoamino acids detected by ninhydrin staining are circled with dots.
Taken together, our data clearly show a physical association of p59fYn with FceRII.
DISCUSSION In this work, we have demonstrated that FceRII/CD23 is capable of transducing an activation signal into lymphocytes. Since IL-2R/p55 induction was seen not only in YTSER cells but also in activated B cells, FceRII may elicit activation of various lymphocyte lineages. Furthermore, the in vitro kinase assays, which most likely utilize the autophosphorylating property of PTK, allowed us to prove that FceRII
Immunology: Sugie et al. A 92.569-
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reflect the indirect fashion ofassociation between a src family PTK and a receptor molecule. All src family PTKs tested have an amino-terminal myristoyl modification, which is essential for anchoring the molecules at the plasma membrane and for their biological functions. Recent reports demonstrated the presence of a membrane protein, p32, which binds p60VSrC (31, 32). We speculate that a membranebound molecule such as p32 might be involved in connecting PTK with its associated receptor.
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We thank Dr. K. Ishizaka for valuable advice; Drs. T. Uchiyama, D. P. Bottaro, H. Wakasugi, and F. Matsuda for the generous gift of cells and reagents; and Miss R. Kasahara and Miss N. Hasegawa for assistance. This work was supported by a Grant-in-Aid for Scientific Research and Special Project Research Cancer Bioscience from the Ministry of Education, Science and Culture of Japan and the Life Science Research Project of Institute of Physical and Chemical Research (RIKEN).
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FIG. 4. Identification of FceRII/CD23-associated PTK of YTSER to p59fYn. (A) Immunoprecipitates with H107 (lane 1), antip5gfyn (lane 2), or anti-p56lck (lane 3) from YTSER were subjected to in vitro kinase assays. After separation on NaDodSO4/polyacrylamide gels, 32P-labeled proteins of 56-59 kDa were treated with 0.5 ,mg of protease V8 per ml and analyzed by 12.5% NaDodSO4/ polyacrylamide gel electrophoresis. (B) Phosphotyrosine-containing proteins affinity-purified from YTSER by using PY20 were preabsorbed with anti-p59fyn (lanes 1 and 3) or anti-p56Ick (lanes 2 and 4) and then immunoprecipitated with H107 mAb (lanes 1 and 2) or control IgG2b (lanes 3 and 4) followed by in vitro kinase assays and 10%6- NaDodSO4/polyacrylamide gel electrophoresis. The arrow indicates the p59 band.
transfected into YT cells was associated with p59fyn. Athough it is not unlikely that only a small fraction of p59fYn is associated with FceRII, the kinase might be involved in the signal transduction through FceRII. Studies are necessary to determine whether the stimulation through FceRII regulates the kinase activity of p59fyn. There are two types of human FceRII (FceRIIa and FceRIIb) that differ only at the aminoterminal cytoplasmic regions (30). The cDNA employed in the present study codes for FcERIIa. Experiments to examine whether FcERIIb is also associated with PTK are necessary. Since parental YT cells did not express FcERII on their, surface unless stimulated with recombinant interleukin 1(, one might wonder that the YTSER transfectant, employed in the present study, provided a rather artificial experimental system and that the FceRII-mediated activation seen in the cells may not represent the physiological mechanism. However, it is of note that the transfection constructed an unequivocally active signal transduction system. Indeed, H107 anti-FceRII mAb had growth-promoting activities as well as IL-2R/p55-inducing activity on YTSER and 3B6 cells (data not shown). An important question is whether PTK is associated with FceRII in B cells. Recent experiments in our laboratory demonstrated an association between FceRII on EBV-transformed B cells and a PTK that was apparently different from src family PTKs such as p59fyn or p56lck. An association of p59fs" with TCR was recently documented in mouse T lymphocytes (24). Although NK-like YT cells had no rearrangement or expression of a, A, y, and a chain genes of TCR complex (J.Y., unpublished observations), the transfected FcERII gene product was found to interact with the endogenous p59fYn molecule of YT cells. It is thus evident that p59fyn can be associated not only with TCR but also with other receptors such as FceRII. This might
1. Yodoi, J. & Ishizaka, K. (1979) J. Immunol. 122, 2577-2583. 2. Capron, A., Dessaint, J. P., Capron, M., Joseph, M., Ameisen, J. C. & Tonnel, A. B. (1986) Immunol. Today 7, 15-18. 3. Bonnefoy, J.-Y., Aubry, J.-P., Peronne, C., Wojdenes, J. & Banchereau, J. (1987) J. Immunol. 138, 2970-2978. 4. Yukawa, K., Kikutani, H., Owaki, H., Yamasaki, K., Yokota, A., Nakamura, H., Barsumian, E. L., Hardy, R. R., Suemura, M. & Kishimoto, T. (1987) J. Immunol. 138, 2576-2580. 5. Kintner, C. & Sugden, B. (1981) Nature (London) 294, 458-460. 6. Thorley-Lawson, D. A., Nadler, L. M., Bhan, A. K. & Schooley, R. T. (1985) J. Immunol. 134, 3007-3012. 7. Gordon, J., Webb, A. J., Walker, L., Guy, G. R. & Rowe, M. (1986) Eur. J. Immunol. 16, 1627-1630. 8. Luo, H., Hofstetter, H., Banchereau, J. & Delespesse, G. (1991) J. Immunol. 146, 2122-2129. 9. Sarfati, M., Nutman, T. B., Suter, U., Hofstetter, H. & Delespesse, G. (1987) J. Immunol. 139, 4055-4060. 10. Kawabe, T., Takami, M., Hosoda, M., Maeda, Y., Sato, S., Mayumi, M., Mikawa, H., Arai, K. & Yodoi, J. (1988)J. Immunol. 141,1376-1382. 11. Hosoda, M., Makino, S., Kawabe, T., Maeda, Y., Satoh, S., Takami, M., Mayumi, M., Arai, K., Saitoh, H. & Yodoi, J. (1989) J. Immunol. 143, 147-152. 12. Yodoi, J., Hosoda, M., Takami, M. & Kawabe, T. (1989) Chem. Immunol. 47, 106-127. 13. Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M. S., Minato, N., Tsudo, M., Uchiyama, T. & Maeda, M. (1985) J. Immunol. 134, 1623-1630. 14. Giorda, R., Rudert, W. A., Vavassori, C., Chambers, W. H., Hiserodt, J. C. & Trucco, M. (1990) Science 249, 1298-1300. 15. Kolb, J.-P., Renard, D., Dugas, B., Genot, E., Petit-Koskas, E., Sarfati, M., Delespesse, G. & Poggioli, J. (1990) J. Immunol. 145, 429-437. 16. Meisenhelder, J., Suh, P. G., Rhee, S. G. & Hunter, T. (1989) Cell 57, 1109-1122. 17. Ikuta, K., Takami, M., Kim, C. W., Honjo, T., Miyoshi, T., Tagaya, Y., Kawabe, T. & Yodoi, J. (1987) Proc. Nat!. Acad. Sci. USA 84, 819-823. 18. Teshigawara, K., Maeda, M., Nishino, K., Nikaido, T., Uchiyama, T., Tsudo, M., Wano, Y. & Yodoi, J. (1985) J. Mol. Cell. Immunol. 2,17-26. 19. Shirakawa, F., Tanaka, Y., Eto, S., Suzuki, H., Yodoi, J. & Yamashita, U. (1986) J. Immunol. 137, 551-556. 20. Narumiya, S., Hirata, M., Nanba, T., Nikaido, T., Taniguchi, Y., Tagaya, Y., Okada, M., Mitsuya, H. & Yodoi, J. (1987) Biochem. Biophys. Res. Commun. 143, 753-760. 21. Koyasu, S., Tagaya, Y., Sugie, K., Yonehara, S., Yodoi, J. & Yahara, I. (1991) J. Immunol. 146, 233-238. 22. Yodoi, J. & Uchiyama, T. (1986) Immunol. Rev. 92, 135-156. 23. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. 24. Samelson, L. E., Phillips, A. F., Luong, E. T. & Klausner, R. D. (1990) Proc. Nat!. Acad. Sci. USA 87, 4358-4362. 25. Wakasugi, H., Rimsky, L., Mahe, Y., Kamel, A. M., Fradelizi, D., Tursz, T. & Bertoglio, J. (1987) Proc. Nat!. Acad. Sci. USA 84,804-808. 26. Noro, N., Yoshioka, A., Adachi, M., Yasuda, K., Masuda, T. & Yodoi, J. (1986) J. Immunol. 137, 1258-1263. 27. Kawakami, T., Kawakami, Y., Aaronson, S. A. & Robbins, K. C. (1988) Proc. Nat!. Acad. Sci. USA 85, 1258-1263. 28. KiWakami, T., Pennington, C. Y. & Robbins, K. C. (1986) Mol. Cell. Biol. 6, 4195-4201. 29. Hunter, T. & Sefton, B. M. (1980) Proc. Nat!. Acad. Sci. USA 77, 1311-1315. 30. Yokoti, A., Kikutani, H., Tanaka, T., Sata, R., Barsumian, E. L., Suemura, M. & Kishimoto, T. (1988) Ce! 55, 611-618. 31. Resh, M. D. (1989) Ce!! 58, 281-286. 32. Resh,;M. D. & Ling, H.-P. (1990) Nature (London) 346, 84-86.
Immunology
Fyn tyrosine kinase associated with FceRII/CD23: Possible multiple roles in lymphocyte activation (YT cells/interleukin 2 receptor/pSS)
KATSUJI SUGIE*t, TOSHIAKI
KAWAKAMI0§, YASUHIRO MAEDA*, TAKUMI KAWABE*, ATSUSHI UCHIDAt,
AND JUNJI YODOI*¶ *Department of Biological Responses, Institute for Virus Research, tDepartment of Late Effect Studies, Radiation Biology Center, and tDepartment of Medical Chemistry, Kyoto University Medical School, Sakyo-ku, Kyoto 606-01, Japan
Communicated by Kimishige Ishizaka, July 17, 1991
Expression of low-affinity Fc receptor for IgE ABSTRACT (FceRll), which is identical to the lymphocyte differentiation antigen CD23, is associated with activation of lymphoid cells. The mechanism of signal transduction through FceRll/CD23 was dissected by transfection of cDNA coding for FccRII to the YT human natural killer-like cell line, activation of which was easily detected by the induction of interleukin 2 receptor/p55(Tac). Cross-linking of FceRII/CD23 with H107 antiFceRll monoclonal antibody markedly enhanced interleukin 2 receptor/p55 expression on the YT cells transfected with FceRH cDNA (YTSER cells). Similar induction of interleukin 2 receptor/p55 by the cross-linking of FcERll was observed on an Epstein-Barr virus-transformed B-cell line, 3B6, and fresh leukemic cells isolated from a patient with B-cell chronic lymphoblastic leukemia. Thus FcERll/CD23 provides activation signals not only in YTSER cells but also in activated B cells. A possible involvement of protein-tyrosine kinase in the FcERII-mediated signal transduction was studied. FcERII was physically associated with a src family tyrosine kinase p59" and not with p56'k, which was also found in YT cells. Recently it was reported that p59r-v was associated with T-cell antigen receptor. Our results collectively suggest the multiple functions of p59fyI that may be implicated in FcERll-mediated activation signal in YT cells.
to indicate direct interactions of FceRI1 molecule with GTPbinding proteins. FceRII has no protein-tyrosine kinase (PTK) domain, whereas other receptors such as plateletderived growth factor receptor and epidermal growth factor receptor were reported to phosphorylate the phospholipase C-y chain (16). In an attempt to define the signal transduction mechanism through FceRII, we transfected cDNA encoding FceRIl (17) to YT cells and established the YTSER cell line constitutively expressing the molecule on the cell surface. Interleukin 2 receptor (IL-2R)/p55(Tac) is induced on YT cells by various stimuli, including cytokines (18, 19) and pharmacological agents (20-22). Cross-linking of FceRII on YTSER cells with H107 anti-FceRII mAb resulted in enhanced expression of IL-2R/p55(Tac). Since PTKs are deeply involved in the growth regulation of various cell types, including lymphoid cells (23), we next tested possible involvement of PTK in FceRII-mediated cell activation. The present experiments show that FceRII on YTSER cells is physically associated with a src family PTK p59fyn, which was recently reported to associate with T-cell antigen receptor (TCR) (24). Possible multiple functions of PTK associated with lymphocyte receptors are discussed.
The low-affinity Fc receptor for IgE (FceRII) is involved not only in the regulation of IgE production (1, 2) but also in the activation or transformation of lymphoid cells, particularly of B-cell lineage. Indeed, FceRII is identical to CD23 (3, 4), which is strongly expressed on Epstein-Barr virus (EBV)transformed B lymphoblast (5, 6). Accumulating evidence suggests that cross-linking of FceRII/CD23 results in signal transduction, which regulates B-cell growth (7, 8). However, biological roles of FcERII/CD23 on non-B cells (9-12) are poorly understood. We have recently found that stimulation of the YT human natural killer (NK)-like cell line (13) with recombinant interleukin 1,B induced FcERII/CD23 (K.S. and Y.M., unpublished observations), suggesting a possible role of the molecule in the activation of NK cells. Interestingly, a cell-surface molecule related to NK function has been reported to show a significant homology with the C-type animal lectins such as FceRII/CD23 (14). These findings collectively indicate the important roles of FcERII/CD23 in various kinds of lymphocyte activation, whereas the molecular mechanism involved in the signal transduction through FceRII remains elusive. It was recently reported that antiCD23 monoclonal antibodies (mAbs) induced a rise in intracellular [Ca2+] and inositol phospholipid hydrolysis in human activated B cells (15). However, no evidence has been shown
MATERIALS AND METHODS Cells and Antibodies. 3B6 (25) is an EBV-transformed B-cell line kindly provided by H. Wakasugi (Institute Gustave Roussy, Villejuif, France). HUT-102 is a human T-lymphotropic virus, type I-positive T-cell line. Fresh leukemic cells of B-cell chronic lymphoblastic leukemia (BCLL) were isolated from peripheral blood of a patient by Ficoll-Paque (Pharmacia). All cells were maintained in RPMI 1640 medium (GIBCO) containing 10% heat-inactivated fetal calf serum (Whittaker Bioproducts) and antibiotics (100 units of penicillin per ml and 100 gg of streptomycin per ml). H107 anti-FceRII mouse IgG2b mAb (26) was purified from ascitic fluid by using protein A-Sepharose affinity chromatography. Antisera against p56lck (anti-LCK) and N-terminal residues 29-43 of p59fY (anti-FYN N) were raised in rabbits as described (27). Anti-Tac mAb and rabbit anti-phosphotyrosine antiserum were generously provided by T. Uchiyama (Kyoto University) and D. P. Bottaro (National Cancer Institute), respectively. PY-20 anti-phosphotyrosine mAb was purchased from ICN. Abbreviations: mAb, monoclonal antibody; FceRII, low-affinity Fc receptor for IgE; NK, natural killer; IL-2R, interleukin 2 receptor; PTK, protein-tyrosine kinase; TCR, T-cell antigen receptor; EBV, Epstein-Barr virus; B-CLL, B-cell chronic lymphoblastic leukemia. §Present address: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037. ITo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Transfection of FcERll/CD23 cDNA. The EcoRI-digested fragment of pKCReSER, including simian virus 40 early promoter/enhancer and FceRII cDNA (17), was inserted into the EcoRI site of pSV2neo (pSV2neoSER). Subclones of YT cells stably transformed with pSV2neoSER (YTSER cells) or pSV2neo (YTNEO-A1 and YTNEO-B1) were established as follows. YT cells (5 x 106) were transfected with 10 Ag of pSV2neoSER or pSV2neo at 2000 V and 8-.uF pulse in cuvettes (Sarstedt) by using an electric cell borer (Richo Chemical Research, Kyoto). After preculture in RPMI 1640 medium containing 10%o fetal calf serum for 48 hr. the transformed cells were maintained in complete culture medium with 0.4 mg of G418 per ml (Sigma) for 4 wk. After selection, the cells transfected with pSV2neoSER (YTSER) were positive for FceRII, as determined by flow cytometry, whereas those transfected with pSV2neo (YTNEO-A1 and YTNEO-B1) were negative (Fig. 1). IL-2R/p55(Tac) Induction. Cells were preincubated on ice with 20 Ag of H107 mAb or control mouse IgG2b (Cappel Laboratories) per ml for 30 min, washed, and cultured with or without 10 ug of goat anti-mouse IgG antibodies per ml (Cappel) for 24 hr. IL-2R/p55(Tac) expression was examined by flow cytometry and Northern blot analysis as described elsewhere (10). Immunoblotting Using Anti-Phosphotyrosine Antiserum. Cells (1.5 x 107) were lysed in 1% Nonidet P-40, 20 mM Tris-HCI (pH 7.2), 150 mM NaCl, 0.1% NaN3, 1 mM phenylmethylsulfonyl fluoride (Calbiochem), 3.3 ug of aprotinin per ml (Sigma), and 0.2 mM sodium orthovanadate (Sigma). The lysates were incubated with H107 mAb or control mouse IgG2b, and immunoprecipitates were recovered with the aid of Pansorbin (Calbiochem). Precipitated proteins were separated on a 10%6 NaDodSO4/polyacrylamide gel and transferred to a poly(vinylidene difluoride) sheet, Immobilon (Millipore). The blots were incubated with rabbit anti-phosphotyrosine antiserum, washed, and treated with '251-labeled protein A ('25Iprotein A; ICN) followed by autoradiography. In Vitro Kinase Assays. In vitro kinase assays were performed by a modification of the method described previously 100
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FcERIIa cDNA was inserted into the EcoRI site of pSV2neo vector and transfected by electroporation. Expression of FceRIIa cDNA was regulated by simian virus 40 early promoter/enhancer systems. After selection with G418, pSV2neoSER transfectants (YTSER cells) expressed FcERII/CD23, as determined by flow cytometry, whereas pSV2neo transfectants (YTNEO-A1 and YTNEO-Bl) did not. A representative result is shown from three experiments with similar results.
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(28). In brief, phosphotyrosine-containing proteins were affinity-purified from lysates of unlabeled cells by using PY20 mAb and subjected to immunoprecipitation by H107 mAb or control IgG2b. Immunoprecipitates were incubated at 250C for 10 min in 0.025 ml of 20 mM Tris HCl (pH 7.2)/5 mM MnCl2 containing 10 1Ci of fy-32PJATP (3000 Ci/mmol; 1 Ci = 37 GBq) and analyzed by 10% NaDodSO4/polyacrylamide gel electrophoresis followed by autoradiography. After in vitro kinase assays, 32P-labeled proteins were subjected to phosphoamino acid analysis as described elsewhere (29). Peptide Mapping. The gel slices after in vitro kinase assays were overlaid with 0.5 ,ug of V8 protease per ml (ICN) and electrophoresed on a 12.5% NaDodSO4/polyacrylamide gel. RESULTS To determine whether FcERII expressed on YTSER cells could transmit activation signals, we measured IL-2R/ p55(Tac) expressed on YTSER cells after stimulation with H107 anti-FcERII mAb. When YTSER cells were treated with H107 mAb and then cross-linked by polyclonal goat anti-mouse IgG antibodies, increased expression of IL-2R/ p55 was detected by flow cytometry (Fig. 2A). In contrast, the treatment with H107 mAb alone or H107 mAb followed by the second antibody did not enhance IL-2R/pS5 expression on parental YT cells. Northern blot analysis also showed that the level of IL-2R/pS5 mRNA in YTSER was upregulated by triggering the cells with H107 mAb and the second antibody (Fig. 2B). These results on the YTSER transfectant raised the possibility that the endogenous FceRII/CD23 on lymphoid cells may also transmit a signal for the induction of IL-2R/p55. As shown in Fig. 2A, cross-linking of the endogenous FceRII expressed on the 3B6 EBV-transformed B-cell line resulted in IL-2R/p55 induction. The cross-linking also induced IL2R/p55 significantly on fresh leukemic cells from a patient with B-CLL. These data collectively indicate that endogenous and transfected FceRII/CD23 could transmit the activation signal into the cells. Since protein-tyrosine phosphorylation has been implicated to play a crucial role in the signal transduction through various cell-surface receptors, we next examined whether tyrosine phosphorylation is involved in FceRII-mediated cell activation. To test whether proteins phosphorylated at tyrosine residues are associated with FceRII in YTSER cells, an immunoblot analysis was performed by the use of polyclonal anti-phosphotyrosine antibody. Several tyrosine-phosphorylated proteins were coimmunoprecipitated by H107 mAb from YTSER cells (Fig. 3A). The most prominent phosphoprotein had a relative molecular mass of 59 kDa (p59). No such coimmunoprecipitation was seen with control clones (YTNEO-A1, YTNEO-B1) or parental YT cells. The results indicate that p59 phosphorylated at tyrosine residues was associated with FcERII on YTSER cells. We next performed in vitro kinase assays to ascertain that the CD23/H107 immune complexes have protein kinase activity. Addition of [y-32P]ATP to the H107-precipitated immune complex from YTSER resulted in phosphorylation of a 59-kDa protein (Fig. 3B, lane 1). In contrast, no phosphorylated product was detected in the parental YT cells (lane 2) or in immunoprecipitates from YTSER cell lysates with control IgG2b (lane 3). A phosphoamino acid analysis of the 59-kDa protein phosphorylated in in vitro kinase assays showed that it was phosphorylated exclusively on tyrosine residues (Fig. 3C). It is thus evident that FcERII of YTSER cells is physically associated with a PTK. A similar association between FceRII and PTK was observed in several FcsRII-positive B-lymphoid cell lines (data not shown). The size of the p59 molecule that is associated with FcERII on YTSER cells falls in the range of the src family PTKs.
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FIG. 2. IL-2R/p55 induction through FceRII. (A) Cells were preincubated on ice with 20 fxg of H107 anti-FcERII mAb (solid lines) or control IgG2b (dashed lines) per ml, washed, and cultured with or without 10 pyg of goat anti-mouse IgG per ml for 24 hr. IL-2R/ p55(Tac) expression was estimated by flow cytometry on a linear scale. Gain was adjusted so that Tac-positive populations before stimulation were around 10%o. Only a representative result is shown among three experiments with similar results. (B) YTSER cells were stimulated (as described in A) with medium alone (lane 1), control IgG2b plus anti-mouse IgG (lane 2), H107 (lane 3), or H107 plus anti-mouse IgG (lane 4). IUT102 (lane 5) is a human T-lymphotropic virus, type I-positive T-cell line. Total cellular RNA was analyzed by Northern blotting using 32P-labeled IL-2R/p55 cDNA probe. Homogeneity of the loaded amounts of RNA was verified by hybridization of the same blot with a pseudo-actin probe. kb, Kilobases.
Using the immune complex kinase assays, we have detected p561ck and p59f" in YT cells (K.S. and T.K., unpublished observations). To determine whether the p59 is identical to p56Ick or p59fYn, we performed V8 protease mapping. Immune complexes prepared from YTSER using antibodies directed against FceRII (H107), p56lck, or p59fyf were subjected to in vitro kinase assays and analyzed on a NaDodSO4/polyacrylamide gel. After autoradiography, the bands of phosphoproteins of56-59 kDa were excised from the gel and exposed to V8 protease. Fig. 4A demonstrated that the pattern of bands derived from partial digestion ofthe p59 was almost identical to that of p59fyt but distinct from that of p561ck. To further confirm that the FceRII-associated p59 is identical to p59fyn, YTSER cell lysates were preabsorbed with anti-p59'yn or anti-p56ck plus fixed staphylococcal Cowan I (Pansorbin), and FceRII was immunoprecipitated with H107 mAb. The in vitro kinase assays demonstrated that the p59 was completely depleted by preabsorption with anti-p599Z but not with anti-p56lck (Fig. 4B).
FIG. 3. Association of PTK with FceRII on YTSER. (A) Cells (1.5 x 107) of YTSER (lanes 1 and 2), YTNEO-A1 (lanes 3 and 4), YTNEO-B1 (lanes 5 and 6), and parental YT (lanes 7 and 8) were solubilized in 1% Nonidet P.40, 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 3.3 mg of aprotinin per ml, and 0.2 mM sodium orthovanadate. Proteins immunoprecipitated with H107 mAb (lanes 1, 3, 5, and 7) or control IgG2b (lanes 2, 4, 6,
and 8) were washed in the lysis buffer, separated on 10%1 NaDodSO4/ polyacrylamide gels, transferred, and probed with a rabbit antiphosphotyrosine antibody (provided by D. P. Bottaro)followed by detection with 1251-protein A. The specificity of the anti-phosphotyrosine antibody was confirmed by including p-nitrophenyl phosphate in the incubation with the antibody. No discernible bands were detected (data not shown). A typical autoradiograph is shown (out of three similar experiments). (B) Phosphotyrosine-containing proteins were affinity-purified from YTSER (lanes 1 and 3) or parental YT (lane 2) with anti-phosphotyrosine mAb PY20 and incubated with H107 mAb (lanes 1 and 2) or control IgG2b (lane 3). Immunoprecipitates were subjected to in vitro kinase assays, in which samples were incubated in 20 mM. Tris-HCl (pH 7.2), 5 mM MnCl2, and 10 ,uCi of [y-t32P]ATP and separated on 10%o NaDodSO4/ polyacrylamide gels. (C) After in vitro kinase assays, 32P-labeled p59 from YTSER was subjected to phosphoamino acid analysis. Positions of phosphoamino acids detected by ninhydrin staining are circled with dots.
Taken together, our data clearly show a physical association of p59fYn with FceRII.
DISCUSSION In this work, we have demonstrated that FceRII/CD23 is capable of transducing an activation signal into lymphocytes. Since IL-2R/p55 induction was seen not only in YTSER cells but also in activated B cells, FceRII may elicit activation of various lymphocyte lineages. Furthermore, the in vitro kinase assays, which most likely utilize the autophosphorylating property of PTK, allowed us to prove that FceRII
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reflect the indirect fashion ofassociation between a src family PTK and a receptor molecule. All src family PTKs tested have an amino-terminal myristoyl modification, which is essential for anchoring the molecules at the plasma membrane and for their biological functions. Recent reports demonstrated the presence of a membrane protein, p32, which binds p60VSrC (31, 32). We speculate that a membranebound molecule such as p32 might be involved in connecting PTK with its associated receptor.
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We thank Dr. K. Ishizaka for valuable advice; Drs. T. Uchiyama, D. P. Bottaro, H. Wakasugi, and F. Matsuda for the generous gift of cells and reagents; and Miss R. Kasahara and Miss N. Hasegawa for assistance. This work was supported by a Grant-in-Aid for Scientific Research and Special Project Research Cancer Bioscience from the Ministry of Education, Science and Culture of Japan and the Life Science Research Project of Institute of Physical and Chemical Research (RIKEN).
f. 7
FIG. 4. Identification of FceRII/CD23-associated PTK of YTSER to p59fYn. (A) Immunoprecipitates with H107 (lane 1), antip5gfyn (lane 2), or anti-p56lck (lane 3) from YTSER were subjected to in vitro kinase assays. After separation on NaDodSO4/polyacrylamide gels, 32P-labeled proteins of 56-59 kDa were treated with 0.5 ,mg of protease V8 per ml and analyzed by 12.5% NaDodSO4/ polyacrylamide gel electrophoresis. (B) Phosphotyrosine-containing proteins affinity-purified from YTSER by using PY20 were preabsorbed with anti-p59fyn (lanes 1 and 3) or anti-p56Ick (lanes 2 and 4) and then immunoprecipitated with H107 mAb (lanes 1 and 2) or control IgG2b (lanes 3 and 4) followed by in vitro kinase assays and 10%6- NaDodSO4/polyacrylamide gel electrophoresis. The arrow indicates the p59 band.
transfected into YT cells was associated with p59fyn. Athough it is not unlikely that only a small fraction of p59fYn is associated with FceRII, the kinase might be involved in the signal transduction through FceRII. Studies are necessary to determine whether the stimulation through FceRII regulates the kinase activity of p59fyn. There are two types of human FceRII (FceRIIa and FceRIIb) that differ only at the aminoterminal cytoplasmic regions (30). The cDNA employed in the present study codes for FcERIIa. Experiments to examine whether FcERIIb is also associated with PTK are necessary. Since parental YT cells did not express FcERII on their, surface unless stimulated with recombinant interleukin 1(, one might wonder that the YTSER transfectant, employed in the present study, provided a rather artificial experimental system and that the FceRII-mediated activation seen in the cells may not represent the physiological mechanism. However, it is of note that the transfection constructed an unequivocally active signal transduction system. Indeed, H107 anti-FceRII mAb had growth-promoting activities as well as IL-2R/p55-inducing activity on YTSER and 3B6 cells (data not shown). An important question is whether PTK is associated with FceRII in B cells. Recent experiments in our laboratory demonstrated an association between FceRII on EBV-transformed B cells and a PTK that was apparently different from src family PTKs such as p59fyn or p56lck. An association of p59fs" with TCR was recently documented in mouse T lymphocytes (24). Although NK-like YT cells had no rearrangement or expression of a, A, y, and a chain genes of TCR complex (J.Y., unpublished observations), the transfected FcERII gene product was found to interact with the endogenous p59fYn molecule of YT cells. It is thus evident that p59fyn can be associated not only with TCR but also with other receptors such as FceRII. This might
1. Yodoi, J. & Ishizaka, K. (1979) J. Immunol. 122, 2577-2583. 2. Capron, A., Dessaint, J. P., Capron, M., Joseph, M., Ameisen, J. C. & Tonnel, A. B. (1986) Immunol. Today 7, 15-18. 3. Bonnefoy, J.-Y., Aubry, J.-P., Peronne, C., Wojdenes, J. & Banchereau, J. (1987) J. Immunol. 138, 2970-2978. 4. Yukawa, K., Kikutani, H., Owaki, H., Yamasaki, K., Yokota, A., Nakamura, H., Barsumian, E. L., Hardy, R. R., Suemura, M. & Kishimoto, T. (1987) J. Immunol. 138, 2576-2580. 5. Kintner, C. & Sugden, B. (1981) Nature (London) 294, 458-460. 6. Thorley-Lawson, D. A., Nadler, L. M., Bhan, A. K. & Schooley, R. T. (1985) J. Immunol. 134, 3007-3012. 7. Gordon, J., Webb, A. J., Walker, L., Guy, G. R. & Rowe, M. (1986) Eur. J. Immunol. 16, 1627-1630. 8. Luo, H., Hofstetter, H., Banchereau, J. & Delespesse, G. (1991) J. Immunol. 146, 2122-2129. 9. Sarfati, M., Nutman, T. B., Suter, U., Hofstetter, H. & Delespesse, G. (1987) J. Immunol. 139, 4055-4060. 10. Kawabe, T., Takami, M., Hosoda, M., Maeda, Y., Sato, S., Mayumi, M., Mikawa, H., Arai, K. & Yodoi, J. (1988)J. Immunol. 141,1376-1382. 11. Hosoda, M., Makino, S., Kawabe, T., Maeda, Y., Satoh, S., Takami, M., Mayumi, M., Arai, K., Saitoh, H. & Yodoi, J. (1989) J. Immunol. 143, 147-152. 12. Yodoi, J., Hosoda, M., Takami, M. & Kawabe, T. (1989) Chem. Immunol. 47, 106-127. 13. Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M. S., Minato, N., Tsudo, M., Uchiyama, T. & Maeda, M. (1985) J. Immunol. 134, 1623-1630. 14. Giorda, R., Rudert, W. A., Vavassori, C., Chambers, W. H., Hiserodt, J. C. & Trucco, M. (1990) Science 249, 1298-1300. 15. Kolb, J.-P., Renard, D., Dugas, B., Genot, E., Petit-Koskas, E., Sarfati, M., Delespesse, G. & Poggioli, J. (1990) J. Immunol. 145, 429-437. 16. Meisenhelder, J., Suh, P. G., Rhee, S. G. & Hunter, T. (1989) Cell 57, 1109-1122. 17. Ikuta, K., Takami, M., Kim, C. W., Honjo, T., Miyoshi, T., Tagaya, Y., Kawabe, T. & Yodoi, J. (1987) Proc. Nat!. Acad. Sci. USA 84, 819-823. 18. Teshigawara, K., Maeda, M., Nishino, K., Nikaido, T., Uchiyama, T., Tsudo, M., Wano, Y. & Yodoi, J. (1985) J. Mol. Cell. Immunol. 2,17-26. 19. Shirakawa, F., Tanaka, Y., Eto, S., Suzuki, H., Yodoi, J. & Yamashita, U. (1986) J. Immunol. 137, 551-556. 20. Narumiya, S., Hirata, M., Nanba, T., Nikaido, T., Taniguchi, Y., Tagaya, Y., Okada, M., Mitsuya, H. & Yodoi, J. (1987) Biochem. Biophys. Res. Commun. 143, 753-760. 21. Koyasu, S., Tagaya, Y., Sugie, K., Yonehara, S., Yodoi, J. & Yahara, I. (1991) J. Immunol. 146, 233-238. 22. Yodoi, J. & Uchiyama, T. (1986) Immunol. Rev. 92, 135-156. 23. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. 24. Samelson, L. E., Phillips, A. F., Luong, E. T. & Klausner, R. D. (1990) Proc. Nat!. Acad. Sci. USA 87, 4358-4362. 25. Wakasugi, H., Rimsky, L., Mahe, Y., Kamel, A. M., Fradelizi, D., Tursz, T. & Bertoglio, J. (1987) Proc. Nat!. Acad. Sci. USA 84,804-808. 26. Noro, N., Yoshioka, A., Adachi, M., Yasuda, K., Masuda, T. & Yodoi, J. (1986) J. Immunol. 137, 1258-1263. 27. Kawakami, T., Kawakami, Y., Aaronson, S. A. & Robbins, K. C. (1988) Proc. Nat!. Acad. Sci. USA 85, 1258-1263. 28. KiWakami, T., Pennington, C. Y. & Robbins, K. C. (1986) Mol. Cell. Biol. 6, 4195-4201. 29. Hunter, T. & Sefton, B. M. (1980) Proc. Nat!. Acad. Sci. USA 77, 1311-1315. 30. Yokoti, A., Kikutani, H., Tanaka, T., Sata, R., Barsumian, E. L., Suemura, M. & Kishimoto, T. (1988) Ce! 55, 611-618. 31. Resh, M. D. (1989) Ce!! 58, 281-286. 32. Resh,;M. D. & Ling, H.-P. (1990) Nature (London) 346, 84-86.
