Eur. J. Immunol. 1991. 21: 1967-1970
Increased kinase activity of p56ICkvia CD2 activation pathway
1967
Short paper Silvia Danielianv*, Remi Fagard*, AndrBs AlcoverO, Oreste AcutoO and Siegmund Fischefl Unit6 332, Institut National de la SantC et de la Recherche MBdicale, ICGM* and Laboratoire d'Immunologie MolBculaire, Institut Pasteuro, Paris
The tyrosine kinase activity of p56rckis increased in human Tcells activated via CD2* An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p561ck, a lymphocyte-specificprotein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass.We now demonstrate that the tyrosine kinase activity of p56lCkis increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described.Theseresults suggest that p56lCkmay play an important role in the CD2 activation pathway.
1 Introduction CD2 is a non-polymorphic glycoprotein expressed on all human thymocytes and peripheral T cells. The physiological ligand for CD2 is the lymphocyte function-associated antigen-3 (LFA-3), a widely distributed cell surface glycoprotein [l]. A role for CD2 in Tcell activation has been inferred from the observation that certain pairs of anti-CD2 mAb can induce IL 2-dependent Tcell proliferation [2,3]. Anti-CD2 and anti-TcRKD3antibodies or the corresponding physiologicalligands (LFA-3 and antigen) can synergize in Tcell activation [4, 51.
CD2 [20]; (c) co-immunoprecipitation of p56ICkwith the CD4 or CD8 surface glycoproteins [21,22] and an increase in p56lCkkinease activity by cross-linkingCD4 [23] or CD8 ~41.
We have previously observed a reduced mobility on SDS acrylamide gel and a concomitant hyperphosphorylationof p56lCk15min after triggering the CD2 activation pathway in Jurkat cells. This form contained newly phosphorylated serine residues and showed increased tyrosine phosphorylation [20]. Additional phosphorylation of some serine and tyrosine residues was observed in this form. In this study we address the question of whether the tyrosine kinase activity Tcell triggering via the CD2 pathway induces the same of p56ICkcan be modified directly via the CD2 activation events as those stimulated byTcR/CD3ligands, such as IL 2 pathway. We demonstrate that stimulation of Jurkat T cell secretion [2], increased intracellular [Ca2+][6], production line or human PBL by anti-CD2 mAb induces a rapid of the phosphatidylinositol pathway-related secondary increase in the protein tyrosine kinase activity of p561ck,this messengers [7], opening of voltage-insensitive plasma increased activity takes place before the shift to a higher membrane calcium channels [8] and serine phosphorylation molecular mass and decreases thereafter. of the CD3 y chain [9]. Recently, tyrosine phosphorylation of the CD3 5 chain [lo] and of other unidentified cellular proteins [ll-131 has been detected after activation of 2 Materials and methods human T lymphocytes via CD2, suggesting that CD2mediated stimulation of human T cells can activate protein 2.1 Cells and Ab tyrosine kinases. Jurkat cells, clone 77-6.8 (a gift of Dr. K. A. Smith, Dartmouth Medical School, Hanover, NH) were grown in The protein tyrosine kinase p56lCkis expressed almost RPMI 1640 medium (Gibco, Paisley, Scotland) suppleexclusively in T lymphocytes [14-161 and in several human mented with 10% FCS, 2 m~ L-glutamine, penicillin and Tcell leukemic lines [14, 171. The implication of p56lCkin streptomycin. PBMC were isolated from heparinized T cell activation has been suggested by several authors who venous blood of healthy donors by Ficoll-Hypaque (Pharreported first (a) a reduction of the levels of mRNA for macia, Uppsala, Sweden) density gradient centrifugation. p56lCkupon stimulation with lectin mitogens and phorbol mAb anti-CD2 (anti-Tl11, IgG2b; anti-T112, IgG,; antiesters [17]; (b) a change in gel mobility of p56lck by T113, IgG3; [2], anti-CD3 (2Ad2A2, IgM; [25]) were kindly triggering of mouse or human TcR/CD3 [18-20] or human provided by Dr. E. L. Reinhen, Dana-Farber Cancer Institute, Boston, MA. Anti-p561ckis an antibody directed against a synthetic peptide of 25 amino acids corresponding [I 93741 to the N terminus of the protein [26].
*
Recipient of a fellowship from Association de Recherche sur la Polyarthrite. This research was supported in part by grants from Association Contre le Cancer and from Association FranGaise contre les Myopathies.
Correspondence: Silvia Danielian, Unit6 332, INSERM, 22, rue Mkchain, F-75014 Paris, France 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2.2 Cell activation Cells (5 x lo6)were incubated at 37 "C in medium alone or in the presence of the different stimulating agents. mAb were used at 1: 100 dilution of ascites fluid. PMA (Sigma Chemical Co., St Louis, MO) was used at 10 ng/ml. Incubation times are detailed in the figure legends. 0014-2980/91/0808-1967$3.50+ ,2510
1968
Eur. J. Immunol. 1991. 21: 1967-1970
S. Danielian, R. Fagard, A. Alcover et al.
2.3 Inmunoprecipitation of p S C k After activation, cells were centrifuged for 7 s in a microfuge and lysed for 30 min in 0.5 ml of cold RIPA lysis buffer [20]. Following centrifugation, p56ICkwas immunoprecipitated by incubating the lysate on ice for 1h with specific antibodies (1 : 100) followed by a 30-min incubation on ice with a 10% solution of formalin-fixed Staphylococcus aureus (Calbiochem-Behring, La Jolla, CA). Immune complexes were collected by centrifugation at 4 "Cand were subsequently washed three times with lysis buffer lacking SDS, sodium deoxycholate, EDTA, EGTA and NaF.
1100 4-
,
564 7-
en3 324-
2.4 In vitro kinase reactions Immune complexes of p561ckwere washed once with kinase buffer (100 mM Pipes, pH 7.0, 10 mM MnC12, 1%aprotinh e , 1m~ PMSF) and suspended in a total reaction volume of 15 p1 of kinase buffer containing 1 yg of acid-denatured enolase [27], 4.5 yM of unlabeled ATP and 10 pCi of Y - ~ ~ P - A T(5000 P Ci/mmol; Amersham Int., Amersham, GB). After 10 min at 30°C, reactions were terminated by the addition of modified sample buffer (140 mM Tris-HC1, pH 6.8, 10% glycerol, 2% SDS, 2 mM EDTA, 0.1 mM Na3V04, 20% 2-ME) and proteins were resolved by SDS-PAGE on 8% gels. For in vitro autophosphorylation of p56lCkthe same protocol was used without addition of enolase and unlabeled ATP
Figure 1. CD2 activation increases kinase activity of p561ck. (a) Jurkat cells were incubated for 1min (lanes 1-3 and 5) or 30 s (lane 4) at 37°C alone (lane l), with anti-T1ll (lane 2), with anti-CD3 (lane 3), or with anti-T112 plus anti-T113 (lanes 4 and 5) at concentrations specified in Sect. 2.2. p56ICkwas isolated by immunoprecipitation, incubated with the exogenous substrate enolase in the presence of Y-~~P-ATF' and 4.5 pM of unlabeled ATP, and resolved by SDS-PAGE on an 8% gel. The exposure time was 30 min. en: enolase; p56: ~ 5 6 ' " (b) ~ . Freshly isolated human PBMC were suspended in RPMI 1640 and incubated for 1 min at 37°C alone (lane l), with anti-Tllz plus anti-Tl13 (lane 2), with PMA (lane 3), or with anti-T11, (lane 4) at concentrations specified in Sect. 2.2. The cells were then lysed and immunoprecipitatedp56Ick was labeled with Y-~~P-ATP and resolved on an SDS-10% PAGE. The exposure time was 90 min. The mobilities of molecular mass standards (in kDa) are indicated to the left of the autoradiogram.
3 Results 3.1 CD2 stimulation increases ~56'" kinase activity
T l l l (Fig. 1b, lane 4) or PMA at 10 ng/ml (Fig. 1b, lane 3), did not induce an alteration of p561Ckautophosphorylation.
We examined the effects of antibody-mediated stimulation of CD2 on the enzymatic activity of p561Ck(Fig. 1). To this end we made use of the humanT cell-derived leukemic line Jurkat, which bears a mature Tcell surface phenotype (CD2+, CD3+, CD4+, CD8-, CD45+). Like normal peripheral T cell populations, Jurkat cells can be activated in response to CD2 stimulation with mAb anti412 plus anti-Tl l3[28]. The effects of this treatment on the tyrosine kinase activity of p56ICkwere analyzed by immune-complex kinase assays using p56lCk-specificantiserum (Fig. 1a). There was a rapid and significant increase in the kinase activity of p56ICkin cells stimulated by the simultaneous addition of the anti-Tll2 and anti-T113mAb as measured by autophosphorylation and phosphorylation of enolase, an exogenous substrate (Fig. l a , lanes 4 and 5). On the contrary, the activity induced by anti-Tl11 (Fig. 1a, lane 2), a mAb directed at an additional epitope on the CD2 molecule which alone is unable to activate Tcells, was significantly lower than the activity obtained with the activating combination of anti-CD2. A parallel irnmunoblot using anti-p5Gckshowed no changes in the amount of p56Ick immunoprecipitated, indicating an increase in specific enzymatic activity (data not shown). The anti-CD3 mAb (Fig. 1a, lane 3) had no effect on both autopliosphorylation and on exogenous substrate phosphorylat ion.
To verify that increases in p56lckkinase activity were not specific to the leukemic line Jurkat, human PBMC cells were stimulated for 1 min with mAb anti-CD2 (Fig. 1b). Accordingly, CD2 triggering resulted in an increase in the tyrosine kinase activity of p561ck as measured by in vitro autophosphorylation (Fig. 1b , lane 2). In contrast, anti-
3.2 Time course of p5ackkinase activity after CD2 stimulation To determine the time interval between CD2 triggering and subsequent increase of tyrosine kinase activity of p561Ck, kinetic studies were performed (Figs. 2 and 3). Jurkat cells were incubated with anti-T112 plus anti-Tl13 mAb for different times; the cells were lysed and immunoprecipitates of p56lCkwere tested for their ability to phosphorylate enolase in vitro. The results from autoradiograms and densitometry scanning are shown in Fig. 2. Two-to three fold increased phosphorylation of enolase was detected after 30 s of CD2 stimulation with a plateau between 3 and 10 min and a decrease thereafter (Fig. 2 a and c). In this context, it is interesting to note that as previously demonstrated by us [20], 15 min after stimulation of Jurkat cells by anti-Tl12 plus anti-T113 mAb, p56ICkdisplayed a reduced mobility on SDS-PAGE (Fig. 2 b , lane 3).This modification was associated with newly phosphorylated serine residues and increased phosphorylation of tyrosine residues (Tyr505) as shown by in vivo labeling experiments [20]. Thus, the appearance of this form of p56lCkcorrelates with a decrease of its tyrosine kinase activity as measured by the enolase assay. '
Similarly, kinetic studies performed with human PBMC stimulated with anti-T112 plus anti-T113, showed a rapid (within 30s) two fold increase in the kinase activity of
Eur. J. Immunol. 1991. 21: 1967-1970
Increa sed kinase activity of p56Ickvia CD2 activation pathway
1969
p56-
e n-
7 a
-0
-
0,7
Q
0.6
-?! 0
C
a
-0
0
0,5 1
5
15 60 120
minutes of antLCD2 stlmulatlon
0,4
-c E 0,2
?!
0
0,5
1
3 5 10 15 30
minutes of antl-CD2 stlmulatlon
Figure 2. Time course of p56Ickkinase activity from Jurkat cells stimulated by anti-CD2. (a) Jurkat cells were incubatedalone (lane 1) or with anti-Tllz plus anti-T113at 37 "C for 3 min (lane 2), 10min (lane 3), 15 min (lane 4) or 30 min (lane 5). p56ICkwas immunoprecipitated and incubated with enolase, in the presence of Y-~~P-ATP and 10 p~ of unlabeled ATP (this high concentration explains the incapacity to detect p561Ckauto-phosphorylation).The exposure time was 10 h. en, enolase. (b) Jurkat cells were incubated alone (lane 1)or with anti-Tllzplus anti-T113at 37 "C for 3 min (lane 2), 15 rnin (lane 3) or 30 min (lane 4).The cells were then lysed and immunoprecipitated p561Ckwas labeled with Y-~~P-ATP and resolved by SDS-PAGE on a 10% gel. (c) Jurkat cells were incubated at 37°C for the time indicated with anti-Tl12 plus anti-Tl13 and treated as in (a). The relative phosphorylation level of enolase was obtained from gel scanning (by laser densitometer). The time of stimulation is represented on a logarithmic scale. The results presented here are derived from the combined analysis of three separate experiments.
p56Ick,reaching a plateau between 1 and 15 min (Fig. 3 a and b). The reduced gel mobility form of pWCkwas clearly detectable after 60 min of CD2 triggering (Fig. 3 a, lane 4); again this shift correlated well with the decrease in tyrosine kinase activity of p56ICk.It is noticeable, that in these cells the time of appearance of the shifted form was considerably longer than for Jurkat cells. In addition, we never observed a complete shift of the lower band.These differences could be due to intrinsic heterogenity in PBL subpopulations. These findings show that an increase in the tyrosine kinase activity of p56Ickmeasured by autophosphorylation and by phosphorylation of enolase was induced within seconds
Figure3. Time course of p5dCkkinase activity from PBMC stimulated by anti-CD2. (a) The cells suspended in RPMI 1640 were incubated alone (lane 1) or with anti-Tllz plus anti-Tl13 for 30 s (lane 2), 15 min (lane 3), 60 rnin (lane 4), 120 min (lane 5) or 180 min (lane 6). p56lCkwas isolated by immunoprecipitation, incubated with the exogenous substrate enolase in the presence of Y-~~P-ATP and 4.5 p~ of unlabeled ATF', and resolved by SDSPAGE on an 8% gel.The exposure time was 2 h. en, enolase; p56, p56Ick.(b) PBMC were incubated at 37°C for the time indicated with anti-Tl12plus anti-T113and treated as in Fig. 3a.The relative phosphorylation level of enolase was obtained from gel scanning (by laser densitometer).The time of stimulationis represented on a logarithmic scale. The results presented here are derived from the combined analysis of two separate experiments. Values were normalized with respect to the amount of protein detected by scanning immunoblots of the same experiment.
after CD2 triggering, before the appearance on SDS-PAGE of the higher molecular mass form of p56Ick.
4 Discussion The data reported in the present study demonstrate that human Tcell activation with the anti-CD2 mAb T112 and T113 increased the tyrosine kinase activity of p561Ck.The results from Jurkat cell line and human PBL indicated that the induced tyrosine kinase activity is rapid (within 30 s) after CD2 triggering. In addition, the decrease of p56lCk kinase activity correlated with the appearance of a reduced mobility form of p561Ckon SDS-PAGE.We have previously shown [20] that this form of p56ICk induced by CD2 triggering contained newly phosphorylated serine residues and showed an increased phosphorylation on Tyr505 (the major in vivo tyrosine phosphorylation residue of p56Ick),a site known to down-regulate its tyrosine kinase activity [29, 301. This suggests that the hyperphosphorylated form may display a lower kinase activity. Although, there is no direct
1970
S. Danielian, R. Fagard, A. Alcover et al.
evidence that any of the several phosphorylation events associated with reduced mobility of p561Ckis responsible for the decrease in phosphotransferase activity, we found that the slow migrating form of p56Ickinduced by treatment of cells with vanadate (a phosphatase inhibitor) has a reduced specific activity compared to the form migrating at 56 kDa [311.
Eur. J. Immunol. 1991. 21: 1967-1970
7 Pantaleo, G., Olive, D., Poggi, A., Kozumbo,W. J., Moretta, L. and Moretta, A., Eur. J. Immunol. 1987. 17: 55. 8 Gardner, P., Alcover, A., Kuno, M., Moingeon, I? ,Weyand, C. M., Goronzy, J. and Reinherz, E. L., J. Biol. Chem. 1989.264: 1068. 9 Breitmayer, J. B., Daley, J. F., Levine, H. B. and Schlossman,S. F., J. Immunol. 1987. 139: 2899. 10 Monostori, E., Desai, D., Brown, M. H., Cantrell, D. A. and Crumpton, M. J., J. lmmunol. 1990. 144: 1010. The mechanism of enzymatic activation of p56Ick is not 11 Casnellie, J. E. and Thom, R. E., FEBS Lett. 1990. 261: 331. clearly understood. Reduced p56Ickactivation in vitro [32] and increased phosphorylation on Tyr505[33] are displayed 12 Jin,Y.-J., Kaplan, D. R.,White, M., Spagnoli, G. C., Roberts, T. M. and Reinherz, E. L., J. Irnmunol. 1990. 144: 647. by Tcells lacking the leukocyte common antigen (CD45), Samelson, L. E., Fletcher, M. C., Ledbetter, J. A. and June, C. 13 the major membrane-bound phosphotyrosine phosphatase. H., J. Immunol. 1990. 145: 2448. Indeed, CD45 could directly activate p561ckby dephospho- 14 Fischer, S., Fagard, R., Gacon, G., Genetet, N., Piau, J. F! and rylating Tyr505 [34]. Furthermore, mAb directed at the Blaineau, C., Biochem. Biophys. Res. Commun. 1984. 124: CD45 molecules are strongly co-mitogenic with CD2 but 682. not anti-CD3 mAb [35] and CD45 could be chemically 15 Voronova, A. F., Buss, J. E., Patschinsky, T., Hunter, T. and cross-linked to CD2 in human T lymphocytes [36].ThereSefton, B., Mol.Cell.Bio1. 1985. 4: 2705. fore a hypothetical model may be that binding of mAb to 16 Marth, J. D., Peet, R., Krebs, E. G. andPerlmutter,R. M., Cell 1985. 43: 393. CD2 would alter the localization of CD45 leading to the dephosphorylation of p561Ckat Tyr505and a concomitant 17 Marth, J. D., Lewis, D. B.,Wilson, C. B., Gearn, M. E., Krebs, E. G. and Perlmutter, R. M., EMBO J. 1987. 6: 2727. increase in the kinase activity of p56lCk.However, lack of 18 Marth, J. D., Lewis, D. B., Cooke, M. I?, Mellins, E. D., phosphate on TyrSo5is not sufficient for elevation of the Gearn, M. E., Samelson, L. E.,Wilson, C. B., Miller, A. D. and p56lCktyrosine kinase activity [33,37], which might require Perlmutter, R. M., J. Immunol. 1989. 142: 2430. additional modifications dependent on phosphorylation at 19 Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A. Tyr394,the site of p561ckautophosphorylation [38]. and Bolen, J. B., Mol. Cell. Biol. 1988. 8: 4353. 20 Danielian, S., Fagard, R., Alcover, A., Acuto, 0.and Fischer, S., Eur. J. lmmunol. 1989. 19: 2183. Recent studies have indicated that stimulation of T cells with cross-linked anti-CD4 or antLCD8 mAb results in a 21 Rudd, C.,Trevillyan, J. M., Dasgupta, J. D.,Wong, L. L. and Schlossman, S. F., Proc. Natl. Acad. Sci. USA 1988. 85: net increase of p561ckkinase activity [23, 241. Since p561Ck 5190. binds to defined sites of the intracellular domain of these 22 Veillette, A., Bookman, M. A., Horak, E. M. and Bolen, J. B., receptors [39], and can be co-immunoprecipitated with Cell 1988. 55: 301. them [21, 221, a question raised by this work is whether 23 Veillette, A., Bookman, M. A., Horak, E. M., Samelson, L. E. p56lCkcould also be physically associated with CD2. Unlike and Bolen, J. B., Nature 1989. 338: 257. CD4 or CD8, the cytoplasmicdomain of CD2 [40] does not 24 Luo, K. and Sefton, B. W., Mol. Cell. Biol. 1990. 10: 5305. contain the sequence responsible for the association with 25 Reinherz, E. L., Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Schlossman, S. E, Cell 1982. 30: 735. ~ 5 6 'It~would ~ . therefore be interesting to assess whether CD2 can be co-immunoprecipitated with p56Ickor whether 26 Fischer, S., Fagard, R., Boulet, I. and Gesquibre, J. C., Biochem. Biohys. Res. Commun. 1987.143: 819. an indirect linkage exists between these two molecules. 27 Cooper, J. A., Esch, F. S.,Taylor, S. S. and Hunter,T., J. Biol. Attemps to co-precipitate CD2 with p561Ckhave so far given Chem. 1984. 259: 7835. inconclusive results. Nevertheless, recent studies of immu- 28 Alcover, A., Alberini, C., Acuto, O., Clayton, L. K.,Transy, nolocalization in our laboratory indicated that the localizaC., Spagnoli, G. C., Moingeon, P., Lopez, F! and Reinherz, E. tion of pWckis significantly and specifically changed after L., EMBO J. 1988. 7: 1973. CD2 triggering with the activating combination of anti- 29 Amrein, K. and Sefton, B., Proc. Natl. Acad. Sci. USA 1988. 85: 4247. CD2 mAb (unpublished results). This supports the hypo30 Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F.,Tinker, D. thesis of a functional linkage between p5gCkand CD2. A,, Overell, R. W., Krebs, E. G. and Perlmutter, R. M., Mol. Cell. Biol. 1988. 8: 540. We wish to thank Dr. E. Reinherz for his generous gifts of anti-CD2 31 Danielian, S., Fagard, R., Boulet, I. and Fischer, S., Biochimie mAb. 1990. 72: 315. 32 Mustelin, T., Coggeshall, K. M. and Altman, A., Proc. Natl. Received March 6, 1991. Acad. Sci. USA 1989. 86: 6302. 33 Ostergaard, H. L., Shackelford,T. R., Johnson, H. P., Hyman, R., Sefton, B. M. and Trowbridge, I. S., Proc. Natl. Acad. Sci. 5 References USA 1989. 86: 8959. 1 Dustin, M. L., Sanders, M. E., Shaw, S. and Springer,T. A., J. 34 Mustelin, T. and Altman, A., Oncogene 1990. 5: 809. 35 Schraven, B., Roux, M., Hutmacher, B. and Meuer, S. C., Eur. Exp. Med. 1987. 165: 677. J. lmmunol. 1989. 19: 397. 2 Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, D., Acuto, O., Fitzgerald, K. A., Hodgon, J. C., Protentis, J. F!,Schlossman, S. 36 Schraven, B., Samstag,Y.,Altevogt, I? and Meuer, S. C., Nature 1990. 345: 71. F. and Reinherz, E. L., Cell 1984.36: 897. 3 Brottier, I?, Boumsell, L., Gelin, C. and Bernard, A., J. 37 Veillette, A., Bolen, J. B. and Bookman, M. A,, Mol. Cell. Biol. 1989. 9: 4441. Immunol. 1985. 135: 1624. 4 Yang, S.Y., Rhee, S.,Welte, K. and Dupont, B., J. I.mmunol. 38 Abraham, N. and Veillette, A., Mol. Cell. Biol. 1990. 10: 5197. 1988. 140: 2115. 5 Bierer, B. E., Peterson, A., Gorga, J. C., Herrmann, S. H. and 39 Turner, J. M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M. and Littman, D. R., Cell 1990. 60: 755. Burakoff, S. J., J. Exp. Med. 1988. 168: 1145. 6 Alcover, A. ,We& M. J., Daley, J. F. and Reinherz, E. L., Proc. 40 Seed, B. and Aruffo, A., Proc. Natl. Acad. Sci. USA 1987.84: 3365. Natl. Acad. Sci. USA 1986. 83: 2614.
Increased kinase activity of p56ICkvia CD2 activation pathway
1967
Short paper Silvia Danielianv*, Remi Fagard*, AndrBs AlcoverO, Oreste AcutoO and Siegmund Fischefl Unit6 332, Institut National de la SantC et de la Recherche MBdicale, ICGM* and Laboratoire d'Immunologie MolBculaire, Institut Pasteuro, Paris
The tyrosine kinase activity of p56rckis increased in human Tcells activated via CD2* An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p561ck, a lymphocyte-specificprotein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass.We now demonstrate that the tyrosine kinase activity of p56lCkis increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described.Theseresults suggest that p56lCkmay play an important role in the CD2 activation pathway.
1 Introduction CD2 is a non-polymorphic glycoprotein expressed on all human thymocytes and peripheral T cells. The physiological ligand for CD2 is the lymphocyte function-associated antigen-3 (LFA-3), a widely distributed cell surface glycoprotein [l]. A role for CD2 in Tcell activation has been inferred from the observation that certain pairs of anti-CD2 mAb can induce IL 2-dependent Tcell proliferation [2,3]. Anti-CD2 and anti-TcRKD3antibodies or the corresponding physiologicalligands (LFA-3 and antigen) can synergize in Tcell activation [4, 51.
CD2 [20]; (c) co-immunoprecipitation of p56ICkwith the CD4 or CD8 surface glycoproteins [21,22] and an increase in p56lCkkinease activity by cross-linkingCD4 [23] or CD8 ~41.
We have previously observed a reduced mobility on SDS acrylamide gel and a concomitant hyperphosphorylationof p56lCk15min after triggering the CD2 activation pathway in Jurkat cells. This form contained newly phosphorylated serine residues and showed increased tyrosine phosphorylation [20]. Additional phosphorylation of some serine and tyrosine residues was observed in this form. In this study we address the question of whether the tyrosine kinase activity Tcell triggering via the CD2 pathway induces the same of p56ICkcan be modified directly via the CD2 activation events as those stimulated byTcR/CD3ligands, such as IL 2 pathway. We demonstrate that stimulation of Jurkat T cell secretion [2], increased intracellular [Ca2+][6], production line or human PBL by anti-CD2 mAb induces a rapid of the phosphatidylinositol pathway-related secondary increase in the protein tyrosine kinase activity of p561ck,this messengers [7], opening of voltage-insensitive plasma increased activity takes place before the shift to a higher membrane calcium channels [8] and serine phosphorylation molecular mass and decreases thereafter. of the CD3 y chain [9]. Recently, tyrosine phosphorylation of the CD3 5 chain [lo] and of other unidentified cellular proteins [ll-131 has been detected after activation of 2 Materials and methods human T lymphocytes via CD2, suggesting that CD2mediated stimulation of human T cells can activate protein 2.1 Cells and Ab tyrosine kinases. Jurkat cells, clone 77-6.8 (a gift of Dr. K. A. Smith, Dartmouth Medical School, Hanover, NH) were grown in The protein tyrosine kinase p56lCkis expressed almost RPMI 1640 medium (Gibco, Paisley, Scotland) suppleexclusively in T lymphocytes [14-161 and in several human mented with 10% FCS, 2 m~ L-glutamine, penicillin and Tcell leukemic lines [14, 171. The implication of p56lCkin streptomycin. PBMC were isolated from heparinized T cell activation has been suggested by several authors who venous blood of healthy donors by Ficoll-Hypaque (Pharreported first (a) a reduction of the levels of mRNA for macia, Uppsala, Sweden) density gradient centrifugation. p56lCkupon stimulation with lectin mitogens and phorbol mAb anti-CD2 (anti-Tl11, IgG2b; anti-T112, IgG,; antiesters [17]; (b) a change in gel mobility of p56lck by T113, IgG3; [2], anti-CD3 (2Ad2A2, IgM; [25]) were kindly triggering of mouse or human TcR/CD3 [18-20] or human provided by Dr. E. L. Reinhen, Dana-Farber Cancer Institute, Boston, MA. Anti-p561ckis an antibody directed against a synthetic peptide of 25 amino acids corresponding [I 93741 to the N terminus of the protein [26].
*
Recipient of a fellowship from Association de Recherche sur la Polyarthrite. This research was supported in part by grants from Association Contre le Cancer and from Association FranGaise contre les Myopathies.
Correspondence: Silvia Danielian, Unit6 332, INSERM, 22, rue Mkchain, F-75014 Paris, France 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2.2 Cell activation Cells (5 x lo6)were incubated at 37 "C in medium alone or in the presence of the different stimulating agents. mAb were used at 1: 100 dilution of ascites fluid. PMA (Sigma Chemical Co., St Louis, MO) was used at 10 ng/ml. Incubation times are detailed in the figure legends. 0014-2980/91/0808-1967$3.50+ ,2510
1968
Eur. J. Immunol. 1991. 21: 1967-1970
S. Danielian, R. Fagard, A. Alcover et al.
2.3 Inmunoprecipitation of p S C k After activation, cells were centrifuged for 7 s in a microfuge and lysed for 30 min in 0.5 ml of cold RIPA lysis buffer [20]. Following centrifugation, p56ICkwas immunoprecipitated by incubating the lysate on ice for 1h with specific antibodies (1 : 100) followed by a 30-min incubation on ice with a 10% solution of formalin-fixed Staphylococcus aureus (Calbiochem-Behring, La Jolla, CA). Immune complexes were collected by centrifugation at 4 "Cand were subsequently washed three times with lysis buffer lacking SDS, sodium deoxycholate, EDTA, EGTA and NaF.
1100 4-
,
564 7-
en3 324-
2.4 In vitro kinase reactions Immune complexes of p561ckwere washed once with kinase buffer (100 mM Pipes, pH 7.0, 10 mM MnC12, 1%aprotinh e , 1m~ PMSF) and suspended in a total reaction volume of 15 p1 of kinase buffer containing 1 yg of acid-denatured enolase [27], 4.5 yM of unlabeled ATP and 10 pCi of Y - ~ ~ P - A T(5000 P Ci/mmol; Amersham Int., Amersham, GB). After 10 min at 30°C, reactions were terminated by the addition of modified sample buffer (140 mM Tris-HC1, pH 6.8, 10% glycerol, 2% SDS, 2 mM EDTA, 0.1 mM Na3V04, 20% 2-ME) and proteins were resolved by SDS-PAGE on 8% gels. For in vitro autophosphorylation of p56lCkthe same protocol was used without addition of enolase and unlabeled ATP
Figure 1. CD2 activation increases kinase activity of p561ck. (a) Jurkat cells were incubated for 1min (lanes 1-3 and 5) or 30 s (lane 4) at 37°C alone (lane l), with anti-T1ll (lane 2), with anti-CD3 (lane 3), or with anti-T112 plus anti-T113 (lanes 4 and 5) at concentrations specified in Sect. 2.2. p56ICkwas isolated by immunoprecipitation, incubated with the exogenous substrate enolase in the presence of Y-~~P-ATF' and 4.5 pM of unlabeled ATP, and resolved by SDS-PAGE on an 8% gel. The exposure time was 30 min. en: enolase; p56: ~ 5 6 ' " (b) ~ . Freshly isolated human PBMC were suspended in RPMI 1640 and incubated for 1 min at 37°C alone (lane l), with anti-Tllz plus anti-Tl13 (lane 2), with PMA (lane 3), or with anti-T11, (lane 4) at concentrations specified in Sect. 2.2. The cells were then lysed and immunoprecipitatedp56Ick was labeled with Y-~~P-ATP and resolved on an SDS-10% PAGE. The exposure time was 90 min. The mobilities of molecular mass standards (in kDa) are indicated to the left of the autoradiogram.
3 Results 3.1 CD2 stimulation increases ~56'" kinase activity
T l l l (Fig. 1b, lane 4) or PMA at 10 ng/ml (Fig. 1b, lane 3), did not induce an alteration of p561Ckautophosphorylation.
We examined the effects of antibody-mediated stimulation of CD2 on the enzymatic activity of p561Ck(Fig. 1). To this end we made use of the humanT cell-derived leukemic line Jurkat, which bears a mature Tcell surface phenotype (CD2+, CD3+, CD4+, CD8-, CD45+). Like normal peripheral T cell populations, Jurkat cells can be activated in response to CD2 stimulation with mAb anti412 plus anti-Tl l3[28]. The effects of this treatment on the tyrosine kinase activity of p56ICkwere analyzed by immune-complex kinase assays using p56lCk-specificantiserum (Fig. 1a). There was a rapid and significant increase in the kinase activity of p56ICkin cells stimulated by the simultaneous addition of the anti-Tll2 and anti-T113mAb as measured by autophosphorylation and phosphorylation of enolase, an exogenous substrate (Fig. l a , lanes 4 and 5). On the contrary, the activity induced by anti-Tl11 (Fig. 1a, lane 2), a mAb directed at an additional epitope on the CD2 molecule which alone is unable to activate Tcells, was significantly lower than the activity obtained with the activating combination of anti-CD2. A parallel irnmunoblot using anti-p5Gckshowed no changes in the amount of p56Ick immunoprecipitated, indicating an increase in specific enzymatic activity (data not shown). The anti-CD3 mAb (Fig. 1a, lane 3) had no effect on both autopliosphorylation and on exogenous substrate phosphorylat ion.
To verify that increases in p56lckkinase activity were not specific to the leukemic line Jurkat, human PBMC cells were stimulated for 1 min with mAb anti-CD2 (Fig. 1b). Accordingly, CD2 triggering resulted in an increase in the tyrosine kinase activity of p561ck as measured by in vitro autophosphorylation (Fig. 1b , lane 2). In contrast, anti-
3.2 Time course of p5ackkinase activity after CD2 stimulation To determine the time interval between CD2 triggering and subsequent increase of tyrosine kinase activity of p561Ck, kinetic studies were performed (Figs. 2 and 3). Jurkat cells were incubated with anti-T112 plus anti-Tl13 mAb for different times; the cells were lysed and immunoprecipitates of p56lCkwere tested for their ability to phosphorylate enolase in vitro. The results from autoradiograms and densitometry scanning are shown in Fig. 2. Two-to three fold increased phosphorylation of enolase was detected after 30 s of CD2 stimulation with a plateau between 3 and 10 min and a decrease thereafter (Fig. 2 a and c). In this context, it is interesting to note that as previously demonstrated by us [20], 15 min after stimulation of Jurkat cells by anti-Tl12 plus anti-T113 mAb, p56ICkdisplayed a reduced mobility on SDS-PAGE (Fig. 2 b , lane 3).This modification was associated with newly phosphorylated serine residues and increased phosphorylation of tyrosine residues (Tyr505) as shown by in vivo labeling experiments [20]. Thus, the appearance of this form of p56lCkcorrelates with a decrease of its tyrosine kinase activity as measured by the enolase assay. '
Similarly, kinetic studies performed with human PBMC stimulated with anti-T112 plus anti-T113, showed a rapid (within 30s) two fold increase in the kinase activity of
Eur. J. Immunol. 1991. 21: 1967-1970
Increa sed kinase activity of p56Ickvia CD2 activation pathway
1969
p56-
e n-
7 a
-0
-
0,7
Q
0.6
-?! 0
C
a
-0
0
0,5 1
5
15 60 120
minutes of antLCD2 stlmulatlon
0,4
-c E 0,2
?!
0
0,5
1
3 5 10 15 30
minutes of antl-CD2 stlmulatlon
Figure 2. Time course of p56Ickkinase activity from Jurkat cells stimulated by anti-CD2. (a) Jurkat cells were incubatedalone (lane 1) or with anti-Tllz plus anti-T113at 37 "C for 3 min (lane 2), 10min (lane 3), 15 min (lane 4) or 30 min (lane 5). p56ICkwas immunoprecipitated and incubated with enolase, in the presence of Y-~~P-ATP and 10 p~ of unlabeled ATP (this high concentration explains the incapacity to detect p561Ckauto-phosphorylation).The exposure time was 10 h. en, enolase. (b) Jurkat cells were incubated alone (lane 1)or with anti-Tllzplus anti-T113at 37 "C for 3 min (lane 2), 15 rnin (lane 3) or 30 min (lane 4).The cells were then lysed and immunoprecipitated p561Ckwas labeled with Y-~~P-ATP and resolved by SDS-PAGE on a 10% gel. (c) Jurkat cells were incubated at 37°C for the time indicated with anti-Tl12 plus anti-Tl13 and treated as in (a). The relative phosphorylation level of enolase was obtained from gel scanning (by laser densitometer). The time of stimulation is represented on a logarithmic scale. The results presented here are derived from the combined analysis of three separate experiments.
p56Ick,reaching a plateau between 1 and 15 min (Fig. 3 a and b). The reduced gel mobility form of pWCkwas clearly detectable after 60 min of CD2 triggering (Fig. 3 a, lane 4); again this shift correlated well with the decrease in tyrosine kinase activity of p56ICk.It is noticeable, that in these cells the time of appearance of the shifted form was considerably longer than for Jurkat cells. In addition, we never observed a complete shift of the lower band.These differences could be due to intrinsic heterogenity in PBL subpopulations. These findings show that an increase in the tyrosine kinase activity of p56Ickmeasured by autophosphorylation and by phosphorylation of enolase was induced within seconds
Figure3. Time course of p5dCkkinase activity from PBMC stimulated by anti-CD2. (a) The cells suspended in RPMI 1640 were incubated alone (lane 1) or with anti-Tllz plus anti-Tl13 for 30 s (lane 2), 15 min (lane 3), 60 rnin (lane 4), 120 min (lane 5) or 180 min (lane 6). p56lCkwas isolated by immunoprecipitation, incubated with the exogenous substrate enolase in the presence of Y-~~P-ATP and 4.5 p~ of unlabeled ATF', and resolved by SDSPAGE on an 8% gel.The exposure time was 2 h. en, enolase; p56, p56Ick.(b) PBMC were incubated at 37°C for the time indicated with anti-Tl12plus anti-T113and treated as in Fig. 3a.The relative phosphorylation level of enolase was obtained from gel scanning (by laser densitometer).The time of stimulationis represented on a logarithmic scale. The results presented here are derived from the combined analysis of two separate experiments. Values were normalized with respect to the amount of protein detected by scanning immunoblots of the same experiment.
after CD2 triggering, before the appearance on SDS-PAGE of the higher molecular mass form of p56Ick.
4 Discussion The data reported in the present study demonstrate that human Tcell activation with the anti-CD2 mAb T112 and T113 increased the tyrosine kinase activity of p561Ck.The results from Jurkat cell line and human PBL indicated that the induced tyrosine kinase activity is rapid (within 30 s) after CD2 triggering. In addition, the decrease of p56lCk kinase activity correlated with the appearance of a reduced mobility form of p561Ckon SDS-PAGE.We have previously shown [20] that this form of p56ICk induced by CD2 triggering contained newly phosphorylated serine residues and showed an increased phosphorylation on Tyr505 (the major in vivo tyrosine phosphorylation residue of p56Ick),a site known to down-regulate its tyrosine kinase activity [29, 301. This suggests that the hyperphosphorylated form may display a lower kinase activity. Although, there is no direct
1970
S. Danielian, R. Fagard, A. Alcover et al.
evidence that any of the several phosphorylation events associated with reduced mobility of p561Ckis responsible for the decrease in phosphotransferase activity, we found that the slow migrating form of p56Ickinduced by treatment of cells with vanadate (a phosphatase inhibitor) has a reduced specific activity compared to the form migrating at 56 kDa [311.
Eur. J. Immunol. 1991. 21: 1967-1970
7 Pantaleo, G., Olive, D., Poggi, A., Kozumbo,W. J., Moretta, L. and Moretta, A., Eur. J. Immunol. 1987. 17: 55. 8 Gardner, P., Alcover, A., Kuno, M., Moingeon, I? ,Weyand, C. M., Goronzy, J. and Reinherz, E. L., J. Biol. Chem. 1989.264: 1068. 9 Breitmayer, J. B., Daley, J. F., Levine, H. B. and Schlossman,S. F., J. Immunol. 1987. 139: 2899. 10 Monostori, E., Desai, D., Brown, M. H., Cantrell, D. A. and Crumpton, M. J., J. lmmunol. 1990. 144: 1010. The mechanism of enzymatic activation of p56Ick is not 11 Casnellie, J. E. and Thom, R. E., FEBS Lett. 1990. 261: 331. clearly understood. Reduced p56Ickactivation in vitro [32] and increased phosphorylation on Tyr505[33] are displayed 12 Jin,Y.-J., Kaplan, D. R.,White, M., Spagnoli, G. C., Roberts, T. M. and Reinherz, E. L., J. Irnmunol. 1990. 144: 647. by Tcells lacking the leukocyte common antigen (CD45), Samelson, L. E., Fletcher, M. C., Ledbetter, J. A. and June, C. 13 the major membrane-bound phosphotyrosine phosphatase. H., J. Immunol. 1990. 145: 2448. Indeed, CD45 could directly activate p561ckby dephospho- 14 Fischer, S., Fagard, R., Gacon, G., Genetet, N., Piau, J. F! and rylating Tyr505 [34]. Furthermore, mAb directed at the Blaineau, C., Biochem. Biophys. Res. Commun. 1984. 124: CD45 molecules are strongly co-mitogenic with CD2 but 682. not anti-CD3 mAb [35] and CD45 could be chemically 15 Voronova, A. F., Buss, J. E., Patschinsky, T., Hunter, T. and cross-linked to CD2 in human T lymphocytes [36].ThereSefton, B., Mol.Cell.Bio1. 1985. 4: 2705. fore a hypothetical model may be that binding of mAb to 16 Marth, J. D., Peet, R., Krebs, E. G. andPerlmutter,R. M., Cell 1985. 43: 393. CD2 would alter the localization of CD45 leading to the dephosphorylation of p561Ckat Tyr505and a concomitant 17 Marth, J. D., Lewis, D. B.,Wilson, C. B., Gearn, M. E., Krebs, E. G. and Perlmutter, R. M., EMBO J. 1987. 6: 2727. increase in the kinase activity of p56lCk.However, lack of 18 Marth, J. D., Lewis, D. B., Cooke, M. I?, Mellins, E. D., phosphate on TyrSo5is not sufficient for elevation of the Gearn, M. E., Samelson, L. E.,Wilson, C. B., Miller, A. D. and p56lCktyrosine kinase activity [33,37], which might require Perlmutter, R. M., J. Immunol. 1989. 142: 2430. additional modifications dependent on phosphorylation at 19 Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A. Tyr394,the site of p561ckautophosphorylation [38]. and Bolen, J. B., Mol. Cell. Biol. 1988. 8: 4353. 20 Danielian, S., Fagard, R., Alcover, A., Acuto, 0.and Fischer, S., Eur. J. lmmunol. 1989. 19: 2183. Recent studies have indicated that stimulation of T cells with cross-linked anti-CD4 or antLCD8 mAb results in a 21 Rudd, C.,Trevillyan, J. M., Dasgupta, J. D.,Wong, L. L. and Schlossman, S. F., Proc. Natl. Acad. Sci. USA 1988. 85: net increase of p561ckkinase activity [23, 241. Since p561Ck 5190. binds to defined sites of the intracellular domain of these 22 Veillette, A., Bookman, M. A., Horak, E. M. and Bolen, J. B., receptors [39], and can be co-immunoprecipitated with Cell 1988. 55: 301. them [21, 221, a question raised by this work is whether 23 Veillette, A., Bookman, M. A., Horak, E. M., Samelson, L. E. p56lCkcould also be physically associated with CD2. Unlike and Bolen, J. B., Nature 1989. 338: 257. CD4 or CD8, the cytoplasmicdomain of CD2 [40] does not 24 Luo, K. and Sefton, B. W., Mol. Cell. Biol. 1990. 10: 5305. contain the sequence responsible for the association with 25 Reinherz, E. L., Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Schlossman, S. E, Cell 1982. 30: 735. ~ 5 6 'It~would ~ . therefore be interesting to assess whether CD2 can be co-immunoprecipitated with p56Ickor whether 26 Fischer, S., Fagard, R., Boulet, I. and Gesquibre, J. C., Biochem. Biohys. Res. Commun. 1987.143: 819. an indirect linkage exists between these two molecules. 27 Cooper, J. A., Esch, F. S.,Taylor, S. S. and Hunter,T., J. Biol. Attemps to co-precipitate CD2 with p561Ckhave so far given Chem. 1984. 259: 7835. inconclusive results. Nevertheless, recent studies of immu- 28 Alcover, A., Alberini, C., Acuto, O., Clayton, L. K.,Transy, nolocalization in our laboratory indicated that the localizaC., Spagnoli, G. C., Moingeon, P., Lopez, F! and Reinherz, E. tion of pWckis significantly and specifically changed after L., EMBO J. 1988. 7: 1973. CD2 triggering with the activating combination of anti- 29 Amrein, K. and Sefton, B., Proc. Natl. Acad. Sci. USA 1988. 85: 4247. CD2 mAb (unpublished results). This supports the hypo30 Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F.,Tinker, D. thesis of a functional linkage between p5gCkand CD2. A,, Overell, R. W., Krebs, E. G. and Perlmutter, R. M., Mol. Cell. Biol. 1988. 8: 540. We wish to thank Dr. E. Reinherz for his generous gifts of anti-CD2 31 Danielian, S., Fagard, R., Boulet, I. and Fischer, S., Biochimie mAb. 1990. 72: 315. 32 Mustelin, T., Coggeshall, K. M. and Altman, A., Proc. Natl. Received March 6, 1991. Acad. Sci. USA 1989. 86: 6302. 33 Ostergaard, H. L., Shackelford,T. R., Johnson, H. P., Hyman, R., Sefton, B. M. and Trowbridge, I. S., Proc. Natl. Acad. Sci. 5 References USA 1989. 86: 8959. 1 Dustin, M. L., Sanders, M. E., Shaw, S. and Springer,T. A., J. 34 Mustelin, T. and Altman, A., Oncogene 1990. 5: 809. 35 Schraven, B., Roux, M., Hutmacher, B. and Meuer, S. C., Eur. Exp. Med. 1987. 165: 677. J. lmmunol. 1989. 19: 397. 2 Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, D., Acuto, O., Fitzgerald, K. A., Hodgon, J. C., Protentis, J. F!,Schlossman, S. 36 Schraven, B., Samstag,Y.,Altevogt, I? and Meuer, S. C., Nature 1990. 345: 71. F. and Reinherz, E. L., Cell 1984.36: 897. 3 Brottier, I?, Boumsell, L., Gelin, C. and Bernard, A., J. 37 Veillette, A., Bolen, J. B. and Bookman, M. A,, Mol. Cell. Biol. 1989. 9: 4441. Immunol. 1985. 135: 1624. 4 Yang, S.Y., Rhee, S.,Welte, K. and Dupont, B., J. I.mmunol. 38 Abraham, N. and Veillette, A., Mol. Cell. Biol. 1990. 10: 5197. 1988. 140: 2115. 5 Bierer, B. E., Peterson, A., Gorga, J. C., Herrmann, S. H. and 39 Turner, J. M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M. and Littman, D. R., Cell 1990. 60: 755. Burakoff, S. J., J. Exp. Med. 1988. 168: 1145. 6 Alcover, A. ,We& M. J., Daley, J. F. and Reinherz, E. L., Proc. 40 Seed, B. and Aruffo, A., Proc. Natl. Acad. Sci. USA 1987.84: 3365. Natl. Acad. Sci. USA 1986. 83: 2614.
