signaling pathways in Jurkat T lymphocytes. the CD3 ...

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CD38 ligation results in activation of the Raf-1/mitogen-activated protein kinase and the CD3-zeta/zeta-associated protein-70 signaling pathways in Jurkat T lymphocytes. M Zubiaur, M Izquierdo, C Terhorst, F Malavasi and J Sancho

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 1997 by American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 1997; 159:193-205; ; http://www.jimmunol.org/content/159/1/193

CD38 Ligation Results in Activation of the Raf-l/ Mitogen-Activated Protein Kinase and the CD3-S/S-Associated Protein-70 Signaling Pathways in Jurkat T Lymphocytes' Mercedes Zubiaur,* Manolo Izquierdo,* Cox Terhorst,+ Fabio Malavasi,* and Jaime Sancho2*

H

uman CD38 Ag is a 45-kDa type I1 transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain (1, 2). It is expressed widely in different cell types including T and B lymphocytes (3,4). The tissue distribution of human CD38 appears to be dependent on the differentiation and activation state of the cell: resting and circulating T and B cells are predominantly CD38and activated cells are CD38+. The structural homology between the extracellular domain of CD38 and adenosine diphosphate ribosyl (ADPR)' cyclase had suggested that the function of CD38

*Department of Cellular Biology and Immunology, Instituto de Parasitologia y Biomedicina, Consejo Superior de lnvestigaciones Cientiiicas (CSIC), Granada, Spain; 'Division of Immunology, Bpth Israel Hospital, Harvard Medical School, Boston, M A 021 15; and *Laboratory of Cellular Biology, University of Torino, Torino, Italy, and Institute of Biology and Genetics, University of Ancona, Ancona, Italy Received forpublicationOctober 28, 1997.

24, 1996.AcceptedforpublicationMarch

The costs of publicatlon of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'

This work was supported by Grant FIS 94/0666 from Instituto de Salud Carlos 111, Spain (to [ X ) ; SAF96-01 17 from Interministerial Commission of Science and Technology (CICYT), Spain (to J.S.); CRC 960778 from Norih Atlantic Treaty Organization (NATO), Brussels (to J.S. and C.T.); and Bilateral Project CSlC/CNR 95/96 (to J.S. and F.M.). F.M. was supported by the following agencies: AlRC (Milan, Italy), TELETHON (Rome, Italy), and ACRO (CNR, Rome, Italy), and by the AIDS and TB projects (Higher Institute of Medicine, Rome, Italy). M.Z. and M.I. were supported by a Contract of Reincorporation from the Mlnistry of Education and Science, Spain.

'

Address correspondence and reprint requests to Dr. Jaime Sancho, Department of Cellular Biology and Immunology, lnstituto de Parasitologia y Biomedicina, Consejo Superior delnvestigacionesCientificas,CNentanilla, 11, Cranada 18001, Spain. E-mail address: granada8ipb.csic.es

' Abbrevidtions

used in this paper: ADPR, adenosine diphosphate rlbose; BCR, B cell receptor; Erk, extracellular signal-regulated protein kinase; Garnlg, goat antibody to mouse immunoglobulin; HRP, horseradish peroxidase; ITAM, immunoreceptor tyrosine-based activation motif; MAP, mitogen-activated protein; PdBu, 4a-phorbol 12,13-dibutyrate; PKC, protein kinase C; PLC, phospholipase C; PTK, protein tyrosine kinase; pTyr, phosphotyrosine; PVDF, polyvinylidene difluoride; Ramlg, rabbit anti-mouse immunoglobulin; SH2,Src homology reglon 2; SH3, Src homology region 3; ZAP-70, (-associated protein-70. Copyright 0 1997 by The American Association of lmmunologlsts

could be related to the production of cyclic ADPR, a calciummobilizing agent ( 5 ) . CD38 can also hydrolyze nicotinamide adenine dinucleotide to ADPR and cyclic ADPR to ADPR (6,7), and therefore it can be considered as a multifunctional ectoenzyme (3). CD38 ligation has stimulatory effects on mature T and B lymphocytes (4, 8-10), but inhibits cell growth and induces apoptosis in B cell precursors ( 1 1). AntiLCD38 mAbs also induce transient tyrosine phosphorylation of several intracellular proteins in immature B cells (12), splenic B cells (13), and HL-60 myeloid cells differentiated with retinoic acid (14), including PLC-yl, c-Cbl, p85 subunit of phosphatidylinositol 3'-kinase, Btk, and Syk. However, it is unknown whether similar biochemical signaling events occur in T cells. The TCR is the crucial receptor that controls the activation of T lymphocytes. One of the earliest detectable signaling events after TCR stimulation is the tyrosine phosphorylation of a number of protein substrates, including the invariant subunits of the TCW CD3 complex, PLC-yl, Vav, ZAP-70, Shc, and valosin-containing protein (1.5). However, unlike many growth-factor receptors, the TCR lacks intrinsic PTK activity. One immediate consequence of triggering the TCR involves the induction of PLC-yl-mediated hydrolysis of inositol phospholipids to generate diacylglycerol and inositol polyphosphates, which induce PKC activation and elevate intracellular calcium, respectively. A second PTK-controlled signaling pathway originated from the TCR has been identified, one that couples the TCR to the activation of the Ras/MAP kinase pathway (1 6). When all of these signaling pathways are integrated in a coordinated manner, they lead to full activation (17). Critical regions of the cytoplasmic domain of the CD3 polypeptide chains are the immunoreceptor tyrosine-based activation motifs (ITAMs), three of which are in each CD3 [-chain and one in each of the other CD3 chains (E, y, and 6). These motifs are necessary and sufficient for coupling the TCR to the intracellular signaling machinery, and function by binding key signaling molecules in T cells (15, 18). The ITAMs are thought to function first 0022-1 767/97/$02.00


CD38 ligation with the specific mAb IB4 induced early and late signaling events in Jurkat T cells, as judged by the transient induction of tyrosine phosphorylation of phospholipase C-yl, c-Cbl, {-associated protein (ZAP)-70, Shc, extracellular signalregulated protein kinase-2 (Erk-2) as mitogen-activated protein (MAP) kinase, and increased expression of the activation Ag CD69. Inaddition, CD38 ligation induced Ras-dependent events suchas Erk-2 mobility shift and increased Erk-2 kinaseactivity. Further evidence that Erk-2 activation is regulated by CD38 ligation was obtained indirectly with the observed induction of Raf-1, Lck, and Sos-1 mobility shifts, processes that are believed to be dependent, at least in part, on MAP kinase activation. Using a protein tyrosine kinase inhibitor, herbimycin A, or a protein kinase C inhibitor, Ro-31-8220, we found that the antiCD38-induced Erk-2 activation is both protein tyrosine kinase and protein kinase C dependent. CD38 ligation also resulted in increased CD3-{ tyrosine phosphorylation and its association with ZAP-70. CD38 ligation in a Jurkat Lck-deficient mutant, JCaml, failed to induce substrate tyrosine phosphorylation and activation of Erk-2. These data indicated that in Jurkat T cells, CD38 receptor triggering results in Lck-regulated activation of both Raf-1/MAP kinase and CD3-LJZAP-70/phospholipase C-yl signalingpathways. The Journal of Immunology, 1997, 159: 193-205.

194

Materials and Methods Cell culture The human Jurkat T cell leukemia line (cloneD8) was obtained from wild-type Jurkat cells (clone E6-1: American Type Culture Collection (ATCC), Rockville, MD) by the limiting dilution technique (21). The Lckdefective Jurkat variant, JCam1.6 (22), was obtained from European Collection of Animal Cell Cultures (Salisbury, Wiltshire, U.K.). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (BioWhittaker. Verviers, Belgium), 2 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Growth was at 37°C in a humidified 5% C02/95% air incubator. Cell culture media and reagents were obtained from Life Technologies (Gaithersburg, MD).

Abs and reagents Puritied CD3 mAb OKT3 (IgG2a) was a gift from Dr. Goldstein (OrthoPharmaceutical, Raritan, NJ). Anti-CD38 mAb OKTIO (IgG1) hybridoma was obtained from ATCC and produced as ascites. Anti-CD38 mAb 1B4 (IgG2a) was prepared and purified by affinity chromatography on protein A-Sepharose and HPLC on hydroxyapatite, as described (23). Affinitypurified, FITC-conjugated, F(ab'), fraction of rabbit Ab tomouseIgs (F(ab'), FITC-RamIg) was purchased from DAKO (Glostrup, Denmark). Affinity-purified, F(ab'), fraction of goat Ab to mouse IgG (whole molecule)(F(ab'),Gamlg) was purchased from Cappel (Organon Teknika, Durham, NC). Anti-phosphotyrosine (anti-pTyr) mAb 1G2 coupled to agarose beads ( I G2-agarose) was obtained from Oncogene Research (Calbiochem, Cambridge, MA).Recombinant anti-pTyr Ab coupled to horseradish peroxidase (RC20-HRP), affinity-purified rabbit polyclonal anti-human Shc, and anti-mouse Sosl mAb (raised against the N terminus of mSosl) were obtained from Transduction Laboratories (Lexington, KY). The following affinity-purified rabbit polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): anti-Erk-2, anti-Raf- 1, anti-c-Cbl, and anti-human Sosl (C terminus). Anti-Syk (1373-13) rabbit antiserum was a kind gift from Dr. A. Weiss (Howard Hughes Medical Institute, University of California, San Francisco, CA) (24). Anti-Zap-70 (Zap-4) rabbit antiserum was a kind gift from Dr. S. C. Ley (National Institute for Medical Research, London, U.K.) (25). Anti-CD3-5 antiserum 448 was a gift from Dr. B. Alarc6n (Centro de Biologia Molecular (CBM), Madrid, Spain). The anti-CD3-5 mAb 1D4.1 has been previously described (26). Anti-human Lck (N terminus) antiserum was from Upstate Biotechnology (Lake Placid, NY). The anti-PLC-7-1 polyclonal Ab (C-37) was made by immunizing New Zealand White rabbits with the synthetic peptide ADHFDSRERRAPRRTRVNGD conjugated to soluble keyhole limpet hemocyanin (Sigma Chemical Co.-Aldrich Quimica, S.A., Madrid, Spain), as previously described (27). Affinity-purified goat anti-rabbit IgG (Fc) HRP conjugate, and goat anti-mouse IgG (H + L) HRP conjugate were from Promega Corp. (Madison, WI). Prestained SDS-PAGE standards (broad range) were from Bio-Rad (Hercules, CA). Protein kinase C inhibitor Ro-

BY CD38 LIGATION

31-8220 was purchased from Calbiochem (La Jolla, CA). Recombinant protein A-Sepharose was from Phamacia Biotech (San Cugat, Spain).Protein-kinase inhibitor herbimycin A, 4a-phorbol I2,13-dibutyrate (PdBu), mouse IgG-agarose, and rabbit IgG-agarose were purchased from Sigma Chemical Co.-Aldrich Quimica, S.A. Redivue [y3*P]ATP (3000Ci/mmol) was purchased from Amersham (Amersham, Buckinghamshire, U.K.).

FACS analysis Jurkat cells resuspended at 2 X 10' cells/100 pI in blocking buffer (PBS, 0. I % BSA, and 0.1% sodium azide) were incubated with 5 pg/ml of the OKTIO, or I84 antLCD38 mAbs, or OKT3 antiLCD3 mAb for 30 min at 4°C. Cells were then washed and resuspended in blocking buffer. F(ab'), FITC-Rnmlg was added to the samples to a final dilution of 11200 (vI'v). After 30-min incubation at 4°C in the dark, cells were washed as before and resuspended i n 500 p1 of blocking buffer. Samples were analyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Cell stimulation and preparation of cell lysates Cells were grown up to a density of I06/ml, centrifuged, and serum starved in RPMI + 0.5% FBS for 4 h, washed in PBS, and resuspended at 1 to 2 X IO7 cells per sample or as otherwise indicated, in serum-free RPMI-HEPES medium, at 4°C. CD3and CD38 receptors were bound to 5 pg/107 cells of affinity-puritied anti-CD3 mAb (OKT3), or to 5 pg/107 cells of anti-CD38 mAb (IB4), or incubated with RPMI-HEPES medium alone for I O min on ice. This was followed by cross-linking with 20 pg/107cells of an affinitypurified secondary Ab F(ab'), Gamlg (Cappel) for 10 min on ice. Then the cells were incubated at 37°C for the times indicated (30, 15, 5, and 1 min). Control unstimulated and mock-stimulated cells with secondary Ab F(ab')2 GamIg were incubated at 37°C for 30 min. In addition, in the cases indicated, serum-starved and PBS-washed cells were stimulated as following: cells were resuspended (1-4 X IO7 cells) in serum-free RPMI-HEPES, warmed at 37°C forI0 min. The cells were then stimulated by 5 pg/107 cells of OKT3, and by 5 yg/107 cells of IB4, or incubated with RPMI-HEPES, at 37°C for 3 min. This was followed by cross-linking with 20 pg/107 cells of an affinity-purified secondary Ab F(ab'), Gamlg, or with RPMI-HEPES at 37°C for 2 min. As a control, Jurkat cells were incubated with phorbol ester PdBu ( I 0 0 ng/ml) at 37°C for 10 min. In all of the cases, cells were lysed in 1% Nonidet P-40 lysis buffer (solution A) (20 mM HEPES, pH 7.6; 150 mM NaCI; 50 mM NaF: 1 mM Na,VO,; I mM EGTA; 50 pM phenylarsine oxide; 10 mM iodoacetamide; 1 mM PMSF; and 2 pglml of each of small peptide inhibitors, antipain, chymostatin, leupeptin, and pepstatin), for 15 min on ice. Nuclei were removed by centrifugation at 12,000 X g for 15 min at 4°C. Aliquots of whole cell lysates were stored immediately at -80°C or used for immunoprecipitation with various Abs. Laemmli sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-ME, and 0.025% bromophenol blue) was added to aliquots of whole cell lysates, and the mixtures were heated at 90°C for 5 min.

Immunoprecipitation, SDS-PAGE, and Western blotting Before the immunoprecipitation with the anti-pTyr mAb IG2-agarose. or with some of the affinity-purified specific Abs, lysates were precleared for 1 h at 4°C either with 50 pl of mouse IgG-agarose or with 50 p1 of rabbit IgG-agarose, respectively. In the case of using rabbit antisera, lysates were precleared with normal rabbit serum coupled to protein A in lysis buffer. Precleared lysates were incubated with specific Abs for 2 h to overnight at 4°C. Anti-pTyr immunoprecipitates were washed once with 1% Nonidet P-40 lysis buffer (solution A), twice with 0.1% Nonidet P-40 lysis buffer (solution B), and twice with 0.05% Nonidet P-40 lysis buffer (solution C). The immunoprecipitated tyrosine-phosphorylated proteins were eluted with 50 pl of elution buffer (buffer C with 40 mM phenyl phosphate, pH 7.6) for 30min on ice. Beads were removed by centrifugation in 0.45-pm pore size Micropure separators (Amicon, Beverly, MA). After centrifugation, 3X Laemmli sample buffer was added to elute proteins by heating at 90°C for I O min. Other protein complexes were immunoprecipitated by adding protein A-Sepharose beads and incubating for an additional 90 min at 4°C. Immunoprecipitates were washed as described above. Forty microliters of 3X Laemmli sample buffer, with or without 2-ME, was added to the beads, and the immunoprecipitated proteins were eluted from the protein A-Sepharose by heating at 90°C for I O min. Whole cell lysates and immunoprecipitates were separated by SDSPAGE in 10% (or 12.5% gels), as indicated (mini or slab gels; Hoefer, San Francisco, CA). For Sosl and Lck Western blot analysis, 7 and 8% gels were used, respectively. Proteins were transferred electrophoretically to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA),using a semidry transfer apparatus (Hoefer, San Francisco, CA), in a continuous buffer system (39 mM glycine, 48 mM Tris, and 20%


by binding Src family kinases (19). Receptor aggregation then allows these kinases to phosphorylate both tyrosines within the motifs. Because of its tandem SH2 domains, ZAP-70 then binds to these motifs and becomes tyrosine phosphorylated (15, 18, 19). It has been proposed that tyrosine-phosphorylated ZAP-70 then mediates downstream signaling mainly by recruiting other SH2 domain-containing signaling proteins (19, 20). Little progress hasbeenmadeindelineatingthecascade of events that link CD38 to PTK-dependent signaling pathways in T cells. This lack of information is due in part to thelow or negative CD38 surface expression in resting T cells, which hampers biochemical approaches. To circumvent this problem, we have used the well-described T cell line Jurkat as a model system. This cell line and its mutants have been used successfully to dissect the cascade of signaling events linking the TCR to T cell activation ( 1 5 ) . In this study, we have used Jurkat cells, which express both CD38 and TCWCD3 on the cell surface, and JCaml, a Jurkat variant that is Lck deficient, to compare the CD38-mediated early signaling events with those triggered byTCWCD3 engagement. In the present work, we demonstrate that in Jurkat T cells, CD38 receptor triggering results in the activation of the Raf-l/MAP kinase and the CD3-IIZAP-70IPLC-yI pathways by way of signaling cascades, which both depend on Lck.

MAP KINASEACTIVATION A N D ZAP-70

The Journal of Immunology

195

B

A

p

a

OKT3

184

2nd Ab

2nd Ab

cv

p

+

+

z

a zcv

- 205

CD38

- 118

-

86

- 51 1 2 3 4 5 6 7 8 9101112

- 34

WB: Anti-pTyr FIGURE 1. Engagement of CD38 induces tyrosine phosphorylation of a distinct pattern of substrates in Jurkat T cells. A, Phenotypic analysis of Jurkat T cells. Cells were examined for surface expression of CD38 and CD3. Representative flow-cytometric histograms are shown. Jurkat cells were stained with mAbs specific for CD3 (OKT3)and CD38 (OKTIO), followed by F(ab’), FITC-Rarnlg secondary Ab. The negative controls shown in light lines were stained with the secondary Ab alone. Flow-cytometric data are presented as the logarithm of fluorescence intensity. 6, Representative time course experiment of CD38-induced tyrosine phosphorylation in Jurkat T cells is shown. Cells were stimulated as indicated in Materials and Methods. They were incubated for 10 min on ice with anti-CD3 mAb (OKT3) (lanes 2-5), anti-CD38 rnAb (164)(lanes 7-10), or RPMI-HEPES (lanes 1, 6, 1 1, and 12).This was followed by cross-linking with the secondary Ab F(ab’), Gamlg (lanes 1-5, 7-10, and 12)for 10 min on ice. Time course was conducted at 37OC for the times indicated. Control samples were incubated for 30 min at 37OC (lanes 1, 6, 11, and 12).Western blots of whole cell lysates probed with the anti-pTyr rnAb RC-20-HRP are shown. Molecular mass markers are indicated.

methanol), for I to 2 h, at a constant current of 0.8 mA/cm2. The membranes were blocked by incubating them for 1 h at room temperature to overnight at 4°C in either 1% BSA (anti-pTyr blots) or 1 to 5% nonfat dry milk in washing buffer (10 mM Tris, pH7.4, 1 0 0 mMNaCI,and 0.1% Tween-20). Primary Abs were addedin washing buffer with I % BSA (antipTyr RC2O-HRP blots) or I % dry milk for 1 to 3 h at mom temperature to overnight at 4°C. mAb RC2O-HRP was used at 0.05 to 0.1 pg/ml, antiErk-2 was used at 0.02 to 0.04 pg/ml, and anti-Raf-I and anti-c-Cbl were used at 0.05 pg/ml; anti-Shc and anti-mSosI were used at I pg/ml; antiZAP-70antiserum(ZAP-4).anti-Syk, anti-lck, andanti-PLC-y(C-37) were used at 112,000 to 1/5,000 dilution. Anti-CD3-5 antiserum (448) was used at 1/1O,OOO dilution. Secondary Abs coupled to HRP were added at 0.1 to0.2 pg/ml for I h at mom temperature.Thefilterswerewashed extensively between incubations with washing buffer. Blots were developed by chemiluminescence using the enhanced chemiluminescence (ECL) detectionsystemandthenexposedtoHyperfilm-ECL (Amersham). For reprobing.PVDFmembraneswereincubatedfor 2 X 60 min at room temperature in Stripping buffer (0.2 M glycine, pH2.2, 0.1% SDS, and 1 .O% Tween-20). followed by thorough washes with blocking buffer. MAP kinase enzyme assay Jurkat cells were stimulated as indicated above. Whole cell lysates in solution A were diluted 1/10 in kinase buffer (10 mM Tris. pH 7.4, 150 mM NaCI, 2 mM EGTA, 2 mM DIT, 1 mM Na,VO,,, I mM PMSF. 10 pg/ml aprotinin, and 10 pg/mlleupeptin).The in vitrokinaseassaywasperformed using a BIOTRAK. p4Up44 MAP kinase enzyme assay system (Amersham): 15 p1 of diluted sample or kinase buffer was mixed with 10 pI of provided substrate buffer containing a peptide highly selective for MAP kinase, which contains the sequence PLSmP recognized by the kinase and related to sequences of the epidermal growth-factor receptor. This peptide does not contain other phosphorylation sites(28,29). The reaction started by adding the Mg-[”PIATP buffer containing I pCi of [Y-~’P]ATP, in a final volume of 30 pl. To measure endogenous protein phosphorylation, 15 pl of each sample was assayed without the presence of the substrate peptide. This value (background) was subtracted to the value obtained in the presence of peptide. Reactions performedin duplicate at 30°C for 30 min were terminated by the addition of IO pI of orthophosphoric acid.SampleswereappliedontoWhatmanpaperdiscs that werethen washed three times in I % acetic acid, and twice in distilled water. Indi-

vidual paper discs were countedin a Beckman liquid scintillation counter for ‘,P. lnduction of CD69

Cells were suspended in complete RPMI 1 6 4 0 medium, and the following reagents were added at about 10 pg/ml: IB4 mAb (anti-CD38). CBT3-G mAb (anti-CD3). CB19mAb (anti-CDI9). or completemedium alone. The samples were incubated at 37°C for2 h. The cells were then washed twice andstainedwiththeFITC-conjugatedanti-CD69mAbLeu78(Becton Dickinson) and analyzedby flow cytometry. The inductionof CD69 by the different mAbs is expressed as percentage of positively stained cells.

Results CD38 engagement induces substrate tyrosine phosphorylation and CD69 expression in lurkat T cells We first examined in Jurkat T cells the kinetics of substrate tyrosinephosphorylation after CD38 ligation. D8 clone has been subcloned from wild-type Jurkat T cells to get a variant with high CD38 surface expression (see Fig. I A for FACS analysis). In the experiment depicted in Figure IB, we compared the effect of CD38 or CD3 cross-linkingon substrate tyrosinephosphorylation in these cells. Upon CD38 ligation, at least nine substrates at 150, 120, 80, 70, 65, 6 0 , 56, 43, and 39 kDa became tyrosine phosphorylated (Fig. lB, lunes 7-10). This pattern was different from that observed in cells upon CD3 ligation (Fig. IB, lunes 2-5). The major differences relied in the extent of tyrosine phosphorylation and in thatmoresubstratesbecamesignificantlytyrosine phosphorylated after CD3ligation. As expected, no significant increase in tyrosine phosphorylation was detected after incubation of cells with the F(ab‘), GamIg alone (Fig. IB, lunes I and 12). Noteworthy is that the anti-CD38-induced tyrosine phosphorylation of the 43-kDa protein followed slower kinetics than for other substrates. Thus, it peaked at 5 min after CD38 stimulation (lune 9 ) . whereas for most substrates the tyrosine phosphorylationpeak was


c p43

LOG FLUORESCENCE INTENSITY

196 Table I. Induction of CD69 expression (%) on CD38 or CD3 mAb ligation of lurkat T cells" I64

LIGATION BY CD38

M A P KINASEACTIVATION A N D ZAP-70

CBT3-C

59

A

B Lysates Lysates

Ip Anti-pTyr

BU12h

2

"Cells were incubated with Abs at 10 pp,/ml, 3 7 T , for 2 h. " BU12, anti-CD19 isotype-matched mAb. 1 2 3 4 5

AntLCD38 mAbs induce tyrosine phosphorylation, mobility shift, and activation of Erk-2 TCR or CD3 ligation initiates a cascade o f events that culminate in the phosphorylation and activation o f M A P kinase i n T lymphocytes (31-34). The predominant M A P kinase in T cells i s Erk-2 (35). Tyrosinephosphorylation and reduced mobilityon SDSPAGE of Erk-2 (attributable to threonine phosphorylation) have been associated with activation o f this enzyme (33, 34). The experiment depicted in Figure 2A shows an anti-pTyr blot o f whole cell lysates in which a tyrosine-phosphorylated protein at 43 kDa was only observed in cells stimulated 5 min with either anti-CD3 (Fig. 2 A , lune 3 ) or anti-CD38 (Fig. 2 A , lune 4 ) . or stimulated I O min with PdBu(Fig. 2 A , lune 5 ) , and not in unstimulated or mockstimulated cells(Fig. 2 A , lunes I and 2, respectively). T h i s 43-kDa tyrosine-phosphorylated protein was identified as Erk-2 by immunoprecipitation withan anti-pTyr mAb, followed byWestern blotting analysis with an Erk-2-specific A b (Fig. 2B, lunes 6-10). Erk-2 was readily detectable upon 5-min stimulation with anti(Fig. 2B, CD38 or anti-CD3, or upon 10-min incubation with PdBu lunes 9, 8, and 10, respectively). In contrast, no Erk-2 was ob2 rnin with the served in unstimulated cells, or mock stimulated for

FIGURE 2. A and B, CD38 ligation induces tyrosine phosphorylation, mobility shift, and activation of Erk-2. Cells were stimulated by OKT3 (lane 3), by 184 (lane 4 , or incubated with RPMI-HEPES (lanes 7 and 2) at37OC for 3 min, followed by cross-linking with the secondary Ab F(ab'), Gamlg at 37°C for 2 min (lanes 2-4). (See Materials and Methods for details.) As a control, Jurkatcells were incubated with PdBu (100 ns/ml) at 37°C for 10 min (lane 5). A, Anti-pTyr blot of whole celllysates developed with anti-pTyr mAb RC2O-HRP is shown. The 43-kDa tyrosine-phosphorylated protein was only observed in cells stimulated with anti-CD3 (lane 3), anti-CD38 (lane 41, or PdBu (lane5), and not in unstimulated or mock-stimulated cells (lanes 7 and 2, respectively). Position of molecular mass marker is indicated. B, Tyrosine-phosphorylated proteins were immunoprecipitated with the anti-pTyr mAb 1G2-agarose beads (lanes 6-10). Mouse IgC-agarose was used as a control (lane 7 7 ) . Whole cell lysates and immunoprecipitates were resolved on SDS-PACE (10% gel, reducing conditions), and immunoblotted with anti-Erk-2 Ab. In anti-pTyr immunoprecipitates, Erk-2 is readily detectable upon CD38 ligation (lane 9) or anti-

Lysates

1p:Anti-Erk-2

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3

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pmoles/min/3xl05cells equivalents CD3 ligation (lane 8), or incubation with PdBu (lane 70). In contrast, no Erk-2 was observed in unstimulated cells (lane 6 ) , or mock stimulated with the secondary F(ab'), Gamlg Ab alone (lane 7). C, Triggering of the CD38 receptor activates Erk-2 in JurkatT cells. Cells were stimulated as indicated in Figure 1B. Lysates were immunoprecipitated using a specific anti-Erk-2 polyclonal Ab. Whole cell lysates and immunoprecipitateswere resolved by SDS-PACE (10% gel, nonreducing conditions), and probed with the anti-pTyr mAb RC-20-HRP. A tyrosine-phosphorylated protein of approximately 41 to 44 kDa is seen after CD38 ligation by 184 + F(ab'), Camlg in both lysates (lane 3 ) and anti-Erk-2 immunoprecipitates (lanes 9 and 70). An identical tyrosine-phosphorylated protein wasobserved in cells stimulatedby OKT3 + F(ab'), Camlg, in both lysates (lane 2) and anti-Erk-2 immunoprecipitates (lanes 7 and 8), or in cells stimulated with PdBu (lane 5 ) . D, Filter shown in C was stripped and reprobed with an anti-Erk-2 Ab, revealing identical amounts of immunoprecipitated Erk-2 protein. It is clearly shown that CD38 ligation induces a mobility shift of Erk-2 (lanes 3 and 9 ) . The position of Erk-2 is indicated by an arrow. E, Stimulation of MAP kinase activity in Jurkat T cells. Cells were lysed and MAP kinase activity was determined by the enzyme assay system (BIOTRAK,Amersham).Dataareexpressed as pmol ofphosphate transferred per minute, per 3 X lo5 cell equivalents. The mean f SD for duplicated samples are shown. Representative datafrom three experiments with comparable results.


at 1 min after stimulation (lune 10). These observations prompted us to address whether this 43-kDa phosphoprotein could be the microtubule-associated protein-2 kinase (MAP kinase) Erk-2, and to examine whether Erk-2 may be involvedin the signaling transduction pathways triggered by CD38 ligation (see below). We have also examined late signaling events in Jurkat T cells by analyzing the ability o f anti-CD38 mAb IB4 toinduce cellsurface expression o f the activation A g CD69. This A g has been used as a marker o f functional TCWCD3 complex triggering (22, 30). As shown in Table I,triggering o f CD38 withI B 4 m A induced b CD69 surface expression at comparable levels o f that induced by antiCD3 mAb CBT3-G. Overall, these findings indicate that CD38 ligation is able to induce late as well as early signaling events in Jurkat T cells.

9 1011

6 7 8

WB: Anti-Erk-2

WB: Anti-pTyr


Kinetics of CD38-induced mobility shift of Erk-2 and Raf- 1 kinases

Experiments depicted in Figure 1, B and C, suggested that the kinetics of Erk-2 tyrosine phosphorylation was relatively short in CD38-stimulated cells. To further confirm these data, a Western + F(ab’), GamIg, or with blot from cells treated withIB4 OKT3 + F(ab’), GamIg for various periods of time was probed with an anti-Erk-2 antiserum. The data in Figure 3B show that the time course of the CD38-mediated Erk-2 mobility shift was delayed and more transient as compared with the CD3-induced Erk-2 response. Thus, in anti-CD38-stimulated cells, the shift in the elec-

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B

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time (rnin)

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g 5

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2 3 4 5 6 7 8 9101112 WB: Anti-Erk-2

FIGURE 3. Kinetics of CD38 ligation-induced mobility shift ofRaf-1 and Erk-2 kinases. Cells were left unstimulated (control)or mock stimulatedwithF(ab’), Ccrmlg alone, or cellswerestimulatedwith OKT3 + F(ab’), Ccrmlg, or 184 + F(ab’), Gamlg, as described in Figure 1 B. Lysates were resolved by SDS-PACE (10% pel, reducing conditions), transferredto PVDF membrane, and split in two parts.The upper part of the filter was immunoblotted with anti-Raf-1 polyclonal Ab ( A ) .The lower part of the filter was immunoblotted with anti-Erk-2 polyclonal Ab (6).The positions of Raf-1 and Erk-2 are indicated. Only the appropriate parts of the blot are shown.

trophoretic mobility of Erk-2 was slightly detectable after 1 min of CD38 ligation (lane IO),peaked after 5 min (lane 9), and returned rapidly to basal mobility by 15 min (lane 8).In contrast, in antiCD3-stimulated cells, the shift of Erk-2 was readily detectable after 1 min of exposure to OKT3 (lane 5 ) , with a maximum at 5 min (lane 4 ) and a slow return to basal by 30 min (lane 2). MAP kinase activation results in a prolonged shift in the electrophoretic mobility of Raf-l caused by phosphorylation on serine and threonine residues (36). The capacity of CD38 or CD3 ligation to induce Raf-l mobility shift was therefore tested.To this end, a Westem blot of cell lysates prepared fromcells treated with IB4 + F(ab’), GamIg or with OKT3 + F(ab‘), GamIg wa. probed with an antiRaf-l antiserum. As previously shown by Izquierdo et al. (37). the time course of anti-CD3-induced Raf-l mobility shift was delayedin comparison with the Erk-2 response (Fig. 3, A vs B, lanes 2-5). The a shift in Raf-l mobility, data in Figure 3A also show that IB4 induced but with somewhatslower kinetics than that inducedby OKT3. Thus, while in IB4-treated cells the Raf-l mobility shift was only discernible after 5 min of stimulation, with a peak at 15 min that was maintained even after 30 min (Fig. 3A, lanes 7-10), in OKT3-treated cells the Raf-l mobility shift was maximalat 5 min and was maintained up to the 30-min time point (Fig. 3A, lanes 7-10). Herbimycin A inhibits both the anti-CD38-induced increase in tyrosine phosphorylation of Erk-2 and the reduced electrophoretic mobility of Erk-2

We aimed to address the nature of the pathway that could couple CD38 receptor to Erk-2 activation. To this end, we assessed the


secondary F(ab‘), GamIg Ab alone (Fig. 2B, lanes 6 and 7, respectively). The activation of endogenous Erk-2 can be monitored by Westem blot analysis with an anti-Erk-2-specific Ab. The active Erk-2 has a reduced mobility on SDS-PAGE gels compared with nonphosphorylated inactive Erk-2 (34). Therefore, anti-Erk-2 immunoblotting of whole cell lysates can be used to distinguish between activated or nonactivated Erk-2. Thus, in whole cell lysates from OKT3-, IB4-, and PdBu-treated Jurkat cells, there was observed a reduced electrophoretic mobility of Erk-2 (Fig. 2B, lanes 3-5) as compared with control unstimulated or mock-stimulated cells (Fig. 2B. lanes I and 2). To further document that Erk-2 was tyrosine phosphorylated upon CD38 stimulation, this protein was directly immunoprecipitated with a specific anti-Erk-2 Ab, followed by Western blot analysis with an anti-pTyr mAb. As shown in Figure 2C, in Jurkat cells CD38 ligation by IB4 + F(ab‘)2 GamIg induced tyrosine phosphorylation of Erk-2 (Fig. 2C, lanes 9 and I O ) with slower kinetics than CD3 ligation by OKT3 + F(ab‘), GamIg (Fig. 2C, lanes 7 and 8). As expected, a tyrosine phosphoprotein with identical electrophoretic mobility of immunoprecipitated Erk-2 was observed in whole lysates of Jurkat cells stimulated by OKT3, IB4, or phorbol ester PdBu (Fig. 2C, lanes 2,3, and 5, respectively). This tyrosinephosphorylated protein was not observed in nonstimulated cells (Fig. 2C, lanes I and 6 ) or mock stimulated with the F(ab‘), GamIg alone (Fig. 2C, lanes 4 and 1 1 ) . That the phosphoprotein described above was indeed Erk-2 was confirmed by reprobing the same filter with an anti-Erk-2 Ab (Fig. 2 0 ) . Thus, stimulation of Jurkat cells for 1 min with IB4 + F(ab’), GamIg induced a small mobility shift in the band corresponding to Erk-2 (Fig. 2 0 , compare lane I0 with lane 8, corresponding to I-min stimulation with anti-CD38 or anti-CD3, respectively). However, the electrophoretic retardation of Erk2 after 5 minof CD38 ligation (Fig. 20, lanes 3 and 9) was similar to that induced by 5 min of CD3 ligation (Fig. 2 0 , lanes 2 and 7 ) or I O min of PdBu stimulation (Fig. 20, lane 5 ) . These and the above results suggest that there is correlation between the extent of CD38-induced Erk-2 tyrosine phosphorylation and its mobility shift. The CD38-induced tyrosine phosphorylation and mobility shift of Erk-2 suggested CD38-mediated regulation of MAP kinase activity. To test this effect in Jurkat cells, we directly measured MAP kinase activity in vitro using a synthetic peptide as an Erk-specific substrate (see Murerials and Merhods). Figure 2E shows a comparison of MAP kinase activity detected in lysates from Jurkat T cells stimulated for 5 min with anti-CD38, or for 5 min with antiCD3, or for I O min with PdBu. Data revealed that anti-CD38 ligation stimulated approximately 9- to IO-fold increase in MAP kinase activity as compared with unstimulated cells. This activity was alike to that induced by anti-CD3 ligation ( 1 1- to 12-fold increase) or PdBu treatment (7- to 8-fold increase). These results further demonstrate that there is a CD38-mediated activation of the Erk-2 MAP kinase signaling pathway.

197

MAPKINASE AND ZAP-70 ACTIVATION BY CD38 LIGATION

198

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effect of the Src family-specific PTK inhibitor herbimycin A (38) on the induction of substrate tyrosine phosphorylation by antiCD38 stimulation. Pretreatment of cells with 3 pM of herbimycin A for 24 h markedly reduced both basal (Fig. 4A. lane 6 vs lane I ) and anti-CD38-stimulated level of pTyr-containing proteins, including Erk-2 (Fig. 4A, lane 9 vs lane 4 ) . Likewise, in anti-CD3stimulated cells, the same dose of herbimycin A substantially inhibited tyrosine phosphorylation of most proteins (Fig. 4A, lone 8 vs lane 3 ) . We next examined the effect of herbimycin A on the mobility shift of Erk-2. In anti-CD38-stimulated cells, pretreatment with herbimycin A completely inhibited the mobility shift of Erk-2 (Fig. 4B. lane 6 vs lane 5 ) . whereas in anti-CD3- or PdBu-stimulated cells, the mobility shift was only partially inhibited (Fig. 4H. l n r w 4 vs / m e 3, and lane 9 vs lune 8, respectively). Therefore, these results suggest that PTK activity is required for CD38-induced tyrosine phosphorylation and reduced electrophoretic mobility of Erk-2. The effect of the PKC inhibitor Ro-3 1-8220 on the antiCD38-induced mobility shift of Erk2 kinase

We have shown that a PTK-dependent pathway may couple CD38 to Erk-2 activation. but the data did not rule out the contribution of PKC to this response. To this end, we next examined the effect of the PKC selective inhibitor Ro-31-8220 (39. 40) on the CD38induced mobility shift of Erk-2 MAP kinase. As expected, this compound had no effect on the anti-CD38- or anti-CD3-induced pattern of substrate tyrosine phosphorylation (data not shown). However, Ro-3 1-8220 extensively inhibited the Erk-2 mobility shift induced by CD38 ligation at a dose in which PdBu-induced Erk-2 shift was completely inhibited (Fig. 5, / m e 6 vs l m c . 9). In contrast, in agreement with previously published data (34). Ro31-8220 only caused marginal inhibition of CD3-induced Erk-2 shift (Fig. 5, lane 4 vs lane 3 ) . The above results are consistent with the notion that Erk-2 activation induced by CD38 ligation. but not by CD3 engagement, occurs at least in part through a PKCmediated signaling pathway. CD38 ligation induces tyrosine phosphorylation of Shc and mobility shift of hSos 1

Several groups have demonstrated thatthe major pathway by which TCWCD3-mediated signals are relayed directly to the

PKCi

- - -+-++-+++ 1 2 3 4 5 6 7 8 91011

WB Anti-Erk-2 Effect of the PKC inhibitor (PKCi) Ro 31-8220 on CD38inducedmobility shift Erk-2 in Jurkat T cells.Cellsprepared as described in Figure 2A were incubated for 10 min at 37°C with medium alone (lanes 1-3, 5, and 8 ) . o r with 5 pM of Ro 31-8220 (lanes 4, 6, 7, and 9-1 I ) . Cellswere leftunstimulated (lanes I , 7, antl 70) or stimulated with OKT3 + F(ah'), Cnmlg (lanes 3 and 4) or 184 + F(ab'), Camlg (lanes .5 antl 6 ) or PdBu (lanes 8 .1nd O ) , or mock stimulated with the secondary Ab F(ab')2 Gamlg (lanes 2 antl I I ) , as described in Figur? 2A. In lanes 4, 6, 7, antl 9-77, Ro-31-8220was present throughout the stimulation procedure. Whole cell lysates were resolved by SDS-PAGE (lo%,gel, reducing conditions), and the filter blot was probed with a n anti-Erk-2 polyclonal Ab. F I G U R E 5.

Erk 1/2cascade is through Ras-mediated activation of Raf- 1 kinase (31-33. 37). One proposed mechanism for TCR-mediated Ras activation is the stimulation-induced recruitment of the Ras-guanylnucleotide exchange factor Sos to the cell membrane, whichinvolves the formation of a complex of Sos with adaptor proteins Grb2 and Shc (41). To assess Shc function in CD38 signaling pathways in Jurkat T cells, an anti-pTyr mAb was used to immunoprecipitate tyrosine-phosphorylated proteins from Jurkat cells stimulated for 5 min with anti-CD38 or anti-CD3, or 10 min with PdBu, followed by immunoblotting with anti-Shc Ab. In Figure 6A. it is shown that following anti-CD38 or anti-CD3 mAb stimulation. there was an enhancement in tyrosine phosphorylation of Shc (Fig. 6A. lower panel, l a n e s 9 and 8, respectively). In contrast, no increase in tyrosine phosphorylation of Shc wasseen during PdBu or F(ab'), GamIg Ab treatment (Fig. 6A. lower p m d , lrrnes IO and 7, respectively). These findings prompted usto seek for Sos involvement following anti-CD38 stimulation of Jurkat T cells. Anti-phosphotyrosine immunoprecipitates were examined for the presence of


F I G U R E 4. Effect of PTK inhibitorherbimycin A onCD38-inducedproteintyrosinephosphorylation, and mobility shift of Erk-2 in Jurkat cells. A, Jurkat cells were preincubated for 24 h at 37OC in medium alone (lanes 1-5) or 3 pM of herbimycin A (lanes 6-10). Cellswere then washed in PBS and resuspended in serum-free RPMI-HEPES.The stimulation of the cells was conducted as described in Figure 2A: unstimulated (lanes I and 6 ) or mock-stimulated (lanes 2 and 7 ) ,or cells stimulated with OKT3 (lanes 3 and 8 ) or 184 (lmes 4 and 9 ) , and cross-linked with an affinity-purified secondary Ab F(ab'), Camlg, or cells stimulated withPdBu (lanes 5 and I O ) . Cell lysates were resolved by SDS-PAGE (10"/0 pel,reducing conditions) and probed with anti-pTyr mAb RC20-HRP. Positions of tyrosine-phosphorylated Erk-2 and molecular mass markers are indicated to the left of the figure. 6, Duplicated samplesof whole cell lysates from control and stimulated cells were prepared as described in A. The filter was probedwith anti-Erk-2 polyclonal Ab. Position of Erk-2 is indicated by an arrow.

199


A

Lysates

IP Anti-pTyr d

-

sos 1*

203

-118

1 2 3 4 5

6 7 8 9 1011

WB Anti Shc

Shc

7

-

51

1 2 3 4 5

Lysates

IP Anti-Sos 1

n

8

6 7

8 9 1011

FIGURE 6 . CD38ligation induces tyrosinephosphorylation of Shc, and mobility shiftof hSosl. A, Lysates from Jurkatcells either unstimulated (lanes 7 and 6) or stimulated(lanes 3-5and lanes 8-70) or mock stimulated (lanes 2 and 7 ) ,as described in Figure 2A, were subjected to an anti-pTyr immunoprecipitation with the anti-pTyr mAb 1C2-agarose beads. Lysates and immunoprecipitated tyrosine-phosphorylated proteins were resolved bySDS-PACE (10% gel, reducing conditions) and transferred to PVDF membrane. The filter was split in two parts. The upper part of the filter was probed with a n anti-mSosl (N terminus) mAb (A, upper), and the lower part was probed with an anti-Shc antiserum(A, lower). B, Duplicatedlysates from Jurkat cellseither unstirnulated (lanes 7 and 6) or stimulated (lanes 3-5 and lanes 8-70) or mock stimulated (lanes 2 and 7), as described in A, were subjected to a n anti-hSos1 (C terminus)immunoprecipitation.Immunoprecipitates (lanes 6-70) and whole cell lysates (lanes 7-5) were resolved by SDS-PAGE (7% gel), reducing conditions, transferred to PVDFmembrane, and blotted with an anti-mSosl (N terminus) mAb. Rabbit IgCagarose beads were used as a control (lane 7 7). The positions of Shc and Sosl proteins and molecular mass markers are indicated.

hSosl protein (Fig. 6A, upperpanel, lanes 6-10). Although hSosl is not a tyrosine-phosphorylated protein, it was detected in antipTyr immunoprecipitates from both anti-CD38- and antiCD3-stimulated cells (Fig. 6A, upper panel, lanes 9 and X, respectively), but not from PdBu- or F(ab'), GamIg-stimulated cells (Fig. 6A. lanes IO and 7, respectively). One possibility was that hSosl coimmunoprecipitated as part of an Shc-Grb2-Sos signaling complex in which Shc was tyrosine phosphorylated after CD38 receptor stimulation. In turn, this receptor-induced Shc-Grb2-Sos association may contribute to Ras-dependent activation of MAP kinase. It has been reported that Sos is serinekhreonine phosphorylated in several cell types after stimulation with growth factors and other mitogenic agents and detected as a mobility shift on Western blotting. Furthermore, several studies have observed that either MAP kinase kinase itself or other downstream protein kinases such as MAP kinase may be responsible for Sos phosphorylation (36.4248). The effect of CD38 ligation on Sos mobility shift was therefore examined. To this end, immunoprecipitation withan antihuman Sosl anti-peptide Ab (directed against the C terminus) was

lnvolvement of Lck in CD38 signal transduction pathways in lurkat T cells The CD3 ligation-induced shift in the mobility ofLck has been used as an indicator of biologically active TCWCD3 receptors (49). The activation-induced mobility shift of Lck is likely to reflect phosphorylation by both PKC and Erk-2 (50-52). In Figure 7A, the mobility shift of Lck is apparent upon CD38 cross-linking with the anti-CD38 mAb IB4 plus F(ab'), Gamlg (lane 5). As previously described (49, 53, 54). in anti-CD3- or PdBu-treated cells, Lck mobility shift was readily detected (Fig. 7A, lanes 3 and X, respectively). It is noteworthy that there was an exact coincidence between the occurrence of the post-translational modification of Lck and the mobility shift of Erk-2 (compare Fig. 7A and Fig. 4B). Previous studies demonstrate that herbimycin A treatment of T cells results in decreased recovery of Fyn and Lck tyrosine kinases by increasing their rate of degradation (38). Similarly, in Jurkat T cells, herbimycin A treatment depleted the steady state level of Lck by 90% (Fig. 7A, lanes IO and I 1 vs lanes I and 2 ) . In herbimycin A-treated cells, anti-CD38 or anti-CD3 stimulation did not induce the mobility shift of the remaining Lck (Fig. 7A, lanes 6 and 4, respectively), although PdBu treatment did it (Fig. 7A, lane 9 ) . These results suggest that either CD3- or CD38-induced, but not PdBu-induced, changes in electrophoretic mobility of Lck are PTK dependent. Moreover, these data suggest that Lck is a target for CD38-mediated early signal transduction events. The involvement of Lck in CD38 signaling was reinforced by experiments performed with the Lck-deficient Jurkat variant JCaml. These cells are defective in the TCR-mediated induction of tyrosine phosphorylation due to a lack of Lck tyrosine kinase function (22). Analysis of cell lysates by immunoblotting with antipTyr mAb showed that there was no induction of substrate tyrosine phosphorylation following stimulation of JCaml cells with the antiCD38 mAb IB4 cross-linked with a secondary Ab (Fig, 7C, lane 7). or with the anti-CD3 mAb OKT3 alone (Fig. 7C, lane 3 ) . This was in contrast to the response of the parental Jurkat cell line (Figs. 7C, lanes 6 and 2, respectively). The lack of responsiveness of these cells to CD38 stimulation was not due to low CD38 receptor expression, since these cells had similar amounts of CD38 displayed on the cell surface than Jurkat D8 cells (Fig. 7B). Moreover, JCaml cells were able to transduce some signals if anti-CD3 mAb OKT3 cross-linked with F(ab')2 Gamlg was used for stimulation. Under these conditions, there was a weak induction of tyrosine phosphorylation (Fig. 7C, lane 5) as compared with that observed in parental Jurkat cells (Fig. 7C, lane 4 ) . As shown in Figure 7, C and D,in JCam 1 cells stimulated with IB4 + F(ab'), GamIg (lane 7). or with OKT3 alone (lane 3), there was not induction of Erk-2 tyrosine phosphorylation and its mobility shift. In contrast, in JCaml cells stimulated with OKT3 cross-linked with a secondary Ab (lane 5), or with PdBu (lane 9), there was induction of Erk-2 tyrosine phosphorylation and mobility shift. These results strongly suggest thatLckis required for


B

6 7 8 91011

followed by immunoblotting with anti-mouse Sosl mAb (directed against the N terminus). The anti-Sos immunoblot revealed a mobility shift of hSosl after 5 min of anti-CD38 engagement (Fig. 6B. lane 9 ) . However, the reduced electrophoretic mobility of Sos was more prominent after 5 minof anti-CD3 stimulation or 10-min PdBu treatment (Fig. 6B, lmes X and IO, respectively). Similar results were observed by directly immunoblotting whole cell lysates withthe anti-Sosl mAb (Fig. 68, lunes 3-5). These data further supported that in Jurkat T cells MAP kinase pathway is readily activated upon CD38 ligation.

MAP KINASE AND ZAP-70 ACTIVATION BY CD38 LIGATION

200

A

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1 2 3 4 5 6 7 8 91011 WB Anti-lck

B W

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203

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CD38

I

LOG FLUORESCENCE INTENSITY

*

53 +

Erk-2 -,

1 2 3 4 5 6 7 8 91011

34

-1 2 3 4 5 6 7 8 9 1 0 1 1

WB: Anti-Erk-2

WB: Anti-pTyr FIGURE 7. Involvement ofLck in CD38-mediated signaling.A, The effect of PTK inhibitor herbimycinA on CD38-induced mobility shift ofLck

in Jurkat cells. Whole cell lysates from control and stimulated cells prepared as described in Figure4A were separated under nonreducing conditions bySDS-PAGE (8% gel), and blotted with anti-Lck polyclonal rabbit Ab. The position of Lckis indicated by an arrow. B, Phenotypic analysis of JCaml cells. FACS analysis for cell surface expressionof CD3 (leftpanel) or CD38 (right panel) was performed as in Figure 1A. JCaml cells were stained with mAb specific for CD3 (OKT3)or CD38 (1B4),followed by F(ab'), FITC-Ramlg secondary Ab. C, Failure of the Lck-deficient Jurkat cell variant, JCaml, to respond to anti-CD38 stimulation. Parental Jurkat cells (D8) and JCaml cells (JCam)were stimulated as described in Figure 2A with OKT3 alone (lanes 2 and 3), OKT3 + F(ab'), Gamlg (lanes 4 and 5),184 + F(ab'), Gamlg (lanes 6 and 7), or PdBu (lanes 8 and 9),or mock stimulated with F(ab'), Gamlg alone (lane 7 7). Whole cell lysateswere separated under reducing conditions by SDS-PAGE (10% gel) and immunoblotted with the anti-pTyr mAb RC2O-HRP. The positions of the molecular mass markers and tyrosine-phosphorylated Erk-2 are shown. D, CD38 ligation does not induce Erk-2 mobility shift in JCaml cells. Lower part of filter shown in C was stripped and reprobed with an anti-Erk-2 polyclonal Ab (lane 7 vs 6 ) . The position of Erk-2 is indicated.

CD38-mediated increases in substrate tyrosine phosphorylation and Erk-2 activation. lnvolvement of other effector proteins in CD3Smediated intracellular signaling pathways Previous reports have identified PLC-yl, c-Cbl, p85 phosphatidylinositol 3'-kinase, Btk, and Syk as some of the proteins that result in increased tyrosine phosphorylation upon CD38 ligation in B or myeloid cells (12-14). We sought to identify whether some of those proteins were tyrosine phosphorylated in T cells subsequent

to CD38 engagement. To this end, anti-pTyr mAb lG2-agarose was used to immunoprecipitate tyrosine-phosphorylated proteins from Jurkat cells stimulated for 5 min with anti-CD38, or with anti-CD3, or upon IO-min treatment with PdBu. Samples were then analyzed by immunoblotting with a panel of specific Abs to known effector proteins. As shown in Figure 8, clearly PLC-yl, c-Cbl, and ZAP-70 were tyrosine phosphorylated upon CD38 ligation (Fig. 8, A, B, and D,lane 9, respectively). In contrast, there was no evidence for tyrosine phosphorylation of the Syk kinase (Fig. 8C, lune 9 vs 6 ) . As expected, all substrates, including Syk,


JCAM1.6

U -

The Journal o f Immunology Lysates

PLC-’I

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which was also readily detected in cell lysates from OKT3-stimulated cells ( I t r r ~ c 2, ) . was identified as CD3-5by reprobing this part of the filter with an anti-( antiserum that recognizes both unphosphorylated and phosphorylated CD3-( (Fig. 91)). These results show that CD38 ligation induces CD3-( tyrosine phosphorylation and its association with ZAP-70.

Ip Anti-pTyr

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Kinetics of CD3-j tyrosine phosphoryl~~tion upon CD38 ligation

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WB Anti Zap70 FIGURE 8. Tyrosine phosphorylation o i PLC-yl, c-Cbl, and ZAP-70 by CD38 ligation in Jurkat T cells. Cells (4 X 10’ per sample) were prepared and stimulated as descrilml i n Figure 2 A . Lysates were subjected t o inlnlLlnoprec.il,itdtion with the anti-pTyr mAb 1 C2-agarose beads ( b n e s 6-JO), as tlescrihed in Mafc,ria/s and Mcfhods. Mouse IgC-agarose k a t l s were used as a control ( h e J J ) . Lysates ( / a m I-5) or inlnlunopr~cipitates( l a n e s 6-1 I ) were resolved b y SDS-PACE (1 0% gel, reducing conditions) ancl transferred to PVDF, ancl the filter was split in parts and subjected to serial immunohlotting with polyclonal Abs anti-PLCyl ( A ) ,anti-c-Cbl ( R ) , anti-Syk ( 0 , or anti-ZAP-70 ( D ) . Locations of immunoprecipitated proteins are indicated b y arrows. The position of molecular mass markers is indicated to the right of the figure.

resulted in increased tyrosine phosphorylation upon CD3 ligation (Fig. X, A , R, C, and D , / m e X , respectively), but not upon crosslinking with the secondary F(ab’), Gamlg Ab alone (Fig. X. A , R. C, and D , l m e 7, respectively), or PdBu treatment (Fig. X. A. B, C. and D, lrrrw 10, respectively). Involvement of ZAP-70 and the (-chain of the TCR/CD3 complex in the CD38-mediated intracellular signaling pathways

To confirm that indeed ZAP-70 resulted in increased tyrosine phosphorylation upon CD38 engagement,celllysates from antiCD3X-stimulated cells were directly immunoprecipitated with an anti-ZAP-70antiserum,followed by Western blottinganalysis with anti-pTyrmAb RC-2OH. This was compared with the response to anti-CD3 stimulation. As shown in Figure 9A. a 70-kDa tyrosine-phosphorylated protein was detected in cell lysates ( h e 3 ) or ZAP-70 immunoprecipitates ( h e 7) following anti-CD38 stinlulation. This protein was identified as ZAP-70 by reprobing the same filter with an anti-ZAP-70 antiserum (Fig. 9R, 1trw.s 3 and 7). As expected, a higher increase in ZAP-70 tyrosine phosphorylation occurred upon CD3 ligation (Fig. 9A, 1rrle.s 2 and 6). These results agreed with thoseobtained in Figure 8D analyzingantipTyr immunoprecipitates. As shown in Figure 9C. in ZAP-70 immunoprecipitates from both IB4- and OKT3-stimulatedcells,a21-kDa phosphoprotein was coimmunoprecipitated (Fig. YC, l a r w s 7 and 6). This protein,

Discussion In this study, we have characterized some of the early signaling events triggered by CD38 engagement in JurkatTcells. CD3X ligation induces rapid and transient tyrosine phosphorylation of several cytoplasmic proteins, including PLC-yl, c-Cbl, ZAP-70, Shc. Erk-2 MAP kinase, and the CD3 (-chain. The overall increase in substrate tyrosine phosphorylation induced by CD38 ligation ditfers quantitatively and qualitatively from that induced by TCW CD3 ligation. Thus, more substrates result in tyrosine phosphorylation upon TCWCD3 engagement than upon CD38 ligation. The finding on CD38-induced Erk-2 tyrosine phosphorylation prompted us to further explorethe regulation of Erk-2 MAP kinase by engagement of this receptor. It is known that phosphorylation


C

The results on CD38-inducedZAP-70 tyrosine phosphorylation prompted us to examine the kinetics of CD3-( tyrosine phosphorylation upon CD38 ligation. To this end. CD3-< immunoprecipitates from lysates ofCD3X- o r CD3-stimulated cells were analyzed by Western blotting with an anti-pTyr mAb. Samples were run on SDS-PAGE under nonreducing conditions to better distinguish the different phosphorylated forms ofCD3-(. As shown in Figure IOA. after I min of CD38 cross-linking. most tyrosine-phosphorylated CD3-( remained as a 32-kDa protein, and only a small fraction of i t shifted to a 43-kDa protein (Fig. IOA. h e 12).The 43-kDa band was almost undetectable after 5 min ofCD38 stimulation, whereas the 3 2 kDa decreased in intcnsity more slowly than the 43-kDa band and still was detectable 15 min after CD38 ligation (Fig. IOA. 1 m c s / I and 10). In contrast, CD3 stimulation by OKT3 + F(ab’), Camlg induced a more potent increase i n CD3-( tyrosine phosphorylation, followed by an extremely fast CD3-( dephosphorylation process. Thus, while the extent of CD3-( phosphorylation at I rnin upon CD3 ligation isat least several-fold higher than upon CD38 ligation (Fig. IOA, / m e 8 vs 12). there were no ditferences at IS min after CD3 or CD38 ligation (Fig. IOA, / m e 6 vs 1 0 ) . This may reilect a faster process of CD3-5 phosphorylation and/or dephosphorylation in anti-CD3-stimulated cells as compared with those stimulated with anti-CD38. As shown in Figure IOA, a 70-kDa tyrosine-phosphorylated protein was coimmunoprecipitated in anti-CD3-( immunoprecipitates from OKT3-stimulated cells ( l w ~ Xr ) . A similar 70-kDa phosphoprotein was barely detectable in CD3-5 immunoprecipitates from IB4-stimulated cells (Fig. IOA, ltrrw 12). This 70-kDa phosphoprotein was identified as ZAP-70 by reprobing this part of the filter with an anti-ZAP-70 Ab (Fig. IOB). Note that in both CD3- and CD38-stimulatedcells, the kinetics of ZAP-70 association with CD3-( correlated with the kinetics of CD3-5 tyrosine phosphorylation, with a maximum at l min following stimulation ( l a r w s X and 12, respectively). These results agree with previous observations that in CD3-stimulated cells ZAP-70 binding to CD3-( coincides with increased ZAP-70 tyrosine phosphorylation and kinase activity (55, 56). Therefore, the above results (Figs. 9 and I O ) strongly suggest that CD38 engagement by agonist Abs results in activation of the CD3-(/ZAP-70 signaling pathway.

MAP KINASE AND ZAP-70 ACTIVATION BY CD38 LIGATION

202

A-

FIGURE 9. CD38 ligation induces ZAP-70 ty-

A-

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4 5 6 7 8 9 10 11 1 2 1 3

WE: Anti Zap70 FIGURE 10. Kinetics of anti-CD38-inducedCD3-< tyrosine phosphorylation. A, Cells were stimulated as in Figure 16. Time course was conducted at 37OC by incubation of cells with OKT3+ F(ab'), Camlg (lanes 2 and 5-81, 184 + F(ab'), Gamlg (Ianes 3 and 9-12), or F(ab')? Cumlg alone (lane 13). Cell lysates (lanes 1-3) or anti-CD3-< immunoprecipitates (with mAb 1D4.1)were separated on SDS-PAGE (1 2.5"% acrylamide) under nonreducing conditions and subjected to immunoblotting with the anti-pTyr mAb RCZO-HRP. Positions of molecular mass markers are indicated on the right part of the figure. Positions of tyrosine-phosphorylated ZAP-70 and CD3-[ are indicated. The two bands at 45 and 55 kDa are nonspecific bands. 6, The indicated part offiltershown in A was stripped and reprobed with an anti-ZAP-70 antiserum,

o n both tyrosine and threonine residues regulates Erk-2 kinase activity and inducesa reduced electrophoretic mobility on SDSPAGE (33. 34, 57). We have compared the data with the welldescribedphenomenonobserved in anti-CD.3- or PdBu-treated cells (34). CD38 ligation induces transient Erk-2 tyrosine phosphorylation and mobility shift. but with slower kinetics than that induced by CD3 ligation (Fig. 2C and 3 B ) . Furthermore,enzymatic assays in whole cell lysates indicate that indeed CD38 engagement regulates MAP kinase enzymatic activity (Fig. 2 E ) . Elegant studies from several groups indicate that MAP kinase activation by TCRstimulation is through Ras-mediated recruit-

IP: Anti-Zap70

0

WB: a-hp 70

IP:Anti-Zap70

WE: a-CD3-t;

ment of Raf-l to the plasma membrane, and subsequent activation of MAP kinase kinase (31-33. 37). The ability of the TCRto regulate the RaslMAP kinase pathway depends on F'TK and. to a lesser extent, on PKC activation (28,3 1-34, 57). Therefore, it was of interest to establish the potential role of PTKs and PKC on the CD38-mediated regulation of Erk-2. To this end. we have used thc F'TK inhibitor. herbimycin A. and the PKC inhibitor. Ro-3 1-8220. Both reagents readily inhibit the anti-CD38-induced Erk-2 mobility shift (Figs. 4R and 5 ) . In contrast. the PKC inhibitor. Ro-318220. only marginally inhibits the anti-CD3-induced mobility shift of Erk-2, which confirms previous results obtained with Ro-318425. another specific PKC inhibitor of the same family (34).Our results therefore indicate that regulation of Erk-2 MAP kinase by CD38 engagement involves both PTK- and PKC-dependent mechanisms and suggest that signals generated from CD38 o r TCRKD3 to activate Erk-2 are ditferently regulated. Grb2 links receptorlnonreceptor PTKs to Ras signaling pathway by means of its association with Sos ( 5 8 ) . Shc is another widely expressed adaptor protein that becomes tyrosine phosphorylated in response to avariety o f growth factors and associates with the SH2 domain of Grb2 ( 5 9 ) . Shc has been reported to associate with Grb2lSos and to the phosphorylated CD3 (-chain after TCR stimulation (41).We attempted to determine the Shc involvement as an upstream regulator of CD38-mediated MAP kinase signaling pathway. Evidence is provided that Shc becomes tyrosine phosphorylated upon CD38 engagement (Fig. 6). and that association of hSosl with a tyrosine-phosphorylated protein is induced by CD38 ligation. These findings suggest a model for CD38-mediated Ras activation in which Shc results in increased tyrosine phosphorylation, and subsequently interacts with Grb2 and Sos. However, there are at least three other proteins that undergo tyrosine phosphorylation and associate with Grb2 following TCR stimulation. c-Cbl. Lnk, and pp36-38 (16, 60-62). In this sense, c-Cbl is one of the most prominent substrates that results in increased tyrosine phosphorylation upon CD38 ligation in T cells (Fig. 8 R ) . B cells ( 12). and myeloid cells ( l 4 ) , and it may play a critical role in bridging CD38 to the RaslMAP kinase signaling pathway as well. Further evidence that Erk2 activation is regulated by CD38 ligation was obtained indirectly with the observed induction of the Raf-l and hSosl mobility shifts. in fibroblasts and T cells. both processes are believed to be dependent on growth factor or TCR


rosine phosphorylation antl its association with tyrosine-phosphorylatetl CD3-j. A, Cells were prepared and stimulated as in Figure 2A. Cell lysates (lanes 1-31 or ZAP-70 immunoprecipitates (with ZAP-4 antiserum) (hnes 4 - 7 ) were separated hy SDS-PACE (12.5% gel) under reducingconditions, transferred to PVDF, and probed withthe anti-pTyr mAb RCZO-HRP. The position of ZAP-70is indicated. Only the 86- to 4.5-kDa region is shown. 6, The filter shown in A was strippedandreprobedwith the antiZAP-70 antiserum ZAP-4. C, This panel represents the lower part of filter shown in A. Position of tyrosine-phosphorylated 21 -kDa CD3-< is indicated. D, Filter shown in C was stripped andreprol)etlwith the anti-CD3-< antiserum 448, which recognizes bothphosphorylated antlunphosphorylatetl forms ofCD3-6. Positions ot the molecular mass mdrkers and of 21 and 16-kDa forms o t CD3-< are indicated.

IP:Anti-Zap70

Lysates


weak, although significant increase in tyrosine phosphorylation of CD3-l and association of ZAP-70 (Figs. 9 and 10). Previously it has been shown that in fully TCR-stimulated cells, ZAP-70 activation coincides with both CD3-5 binding and tyrosine phosphorylation of ZAP-70, whereas in partial TCR-mediated signaling, it results in dramatic differences in CD3-l and ZAP-70 tyrosine phosphorylation and ZAP-70 activation (70, 71). Our data on CD38-induced tyrosine phosphorylation of both CD3-5 and ZAP-70 (Figs. 9 and IO) and ZAP-70 recruitment in the proximity of CD3-l strongly suggest that stimulation through the CD38 receptor also induces ZAP-70 activation. In Jurkat T cells, PLC-y1 is tyrosine phosphorylated following CD38 ligation (Fig. 8). As a result, PLC-yl may undergo activation with a subsequent increase in intracellular CaZ+concentration (21) and activation of PKC, which in turn is required for CD38mediated activation of MAP kinase. A protein that may be associated with PLC--yI in CD38-stimulated T cells is Lck, which in TCR-stimulated cells becomes associated with the SH2 domain of PLC-yl (72). Alternatively, ZAP-70 may play a role since PLC-yI has been detected in CD3-[/ZAP-70 complexes from TCR-stimulated Jurkat cells (20). Moreover, the increase in intracellular Ca"+ concentration and increased tyrosine phosphorylation of PLC-yI observed after TCR aggregation, and which is absent in ZAP-70deficient T cells, can be reconstituted in these cells by retroviral mediated transduction with the wild-type ZAP-70 gene (73). CD38 ligation in B cell precursors leads to Syk activation without inducing tyrosine phosphorylation of the PTKs, Lyn, Fyn, and Btk (12), suggesting that CD38-mediated activation of Syk is independent of Src family PTKs. Our results, however, suggest that in T cells CD38 triggering results in activation of both Lck and ZAP-70 PTKs, leading to activation of the MAP kinase and PLC-yI signaling pathways. Moreover, considering the experiments done in JCaml cells, Lck seems to be required for CD38mediated increased ZAP-70 tyrosine phosphorylation. Taken together, the data on B and T cells indicate that CD38-mediated signaling is coupled to PTKs of the SyklZAP-70 family, and suggest that their kinase activity is cell lineage regulated. CD38 receptorlacks intrinsic tyrosinekinase domainsor ITAMs (2).Thus, it islikelythatCD38-mediatedincreased tyrosine phosphorylation involves activation of other signaling components. There is increasing evidence that Src familykinases can associate with a number of cell surface molecules that are notmembers of the Ag receptorfamily,includingCD2, CD23, CD36, IL-2RP-chain, and various glycosylphosphatidylinositol-anchored proteins, some of which also require the presence or the simultaneous ligation of the Ag receptor to promote activation. Regarding CD38,there are conflicting data. In B cell lymphomas,CD38-mediatedsignaling requiresthe coexpression of the BCR complex on the cell surface (74). However, in B cell precursorcell lines, which do not express the BCR or associated proteins Iga and Igp, CD38 does not depend on expression of the BCR for signaling (1 2). From our data in Jurkat T cells, clearly both TCR and CD38 share some signaling capabilitiesas the activation of both Raf-I/Erk-2 and CD3-51 ZAP-70signaling pathwaysnecessary for aproductive functional outcome. Furthermore,theresults on CD38-induced CD3-5 tyrosine phosphorylation and recruitment of ZAP-70 in the proximity of the TCR/CD3 complex suggest afunctional relationship between signals delivered through CD38 and TCR. Additional experiments are i n progress to address the question as to whether CD38 needs some of the TCR-associated molecules to access the intracellular signal transduction machinery.


induction of MAP kinase activity, respectively (36, 42-47). The mobility shift of Sos can be mimicked in vitro by phosphorylation of Sos by MAP kinase. In addition, MAP kinase phosphorylation of Sosl promotes dissociation of SosI to different tyrosine-phosphorylated proteins without altering its ability to bind Grb2 (47, 48). Therefore, and according with this model, CD38-mediated activation of MAP kinases through Ras may result in the uncoupling of the Sos/Grb2 complex from tyrosine kinase substrates without blocking the interaction of Sos with Grb2. The signals induced by CD38 ligation are by themselves sufficient to induce a significant CD69 expression (Table I), and it is likely that a Ras/Raf- I/Erk-2-dependent pathway eventually controls CD38-mediated transcription of CD69. Previous studies analyzing the mechanism of PMA- or TCR-induced up-regulation of CD69 in T cells have indeed demonstrated the involvement of Ras (63) and Raf-l (64), and to a much lesser extent, an increase in intracellular calcium (64). Regarding other late signaling events, it has been reported previously that in peripheral blood T cells, CD38 ligation induces proliferation (8), increased CD25 mRNA transcription (65),and secretion of a distinct pattern of cytokines (65, 66). Taken together, these findings clearly show that in T cells CD38 engagement by anti-CD38 mAbs induces late signaling events that lead to full cell activation. Several members of the Src family of protein tyrosine kinases are involved in the transduction of signals from specific cell surface receptors that are not by themselves tyrosine kinase receptors. It has been shown that Lck plays a critical role in MAP kinase activation through TCWCD3 complex (33). In this study, we have searched for the possible involvement of Lck in CD38-mediated signalingevents. We provide evidence that Lck may act as an intermediate in CD38-induced signaling events. Thus, we have shown that post-translational modification of Lck, which is likely to reflect phosphorylation by both PKC and MAP kinases (50-52), is associated intimately with the circumstances under which Erk-2 phosphorylation and activation (as measured as a mobility shift) take place. Moreover, treatment of Jurkat cells with herbimycin A, a PTK inhibitor selective for Src family kinases, inhibits the CD38- or CD3-mediated electrophoretic shift of Lck, whereas PdBu treatment still induces it, suggesting that CD38-induced PTK activation precedes the activation of PKC. Of interest are the findings that Lck may be associated with MAP kinase. The interaction with MAP kinase suggests that Lck is involved in the complex resulting in the activation of MAP kinase (67). In this sense, JCaml cells, which are defective in Lck function, are also defective in CD38-mediated induction of Erk-2 activation (Fig. 7). One interpretation of these results is that Lck is directly coupled to the CD38 receptor and is responsible for the increased tyrosine phosphorylation of proteins observed immediately following stimulation of CD38, including Erk-2. However, Lck may play a regulatory role rather than being directly responsible for such increase in tyrosine phosphorylation. Lck could control the activity of another kinase that mediates signaling through CD38. Occupancy of the TCR causes the initial activation of Lck andor Fyn, leading to tyrosine phosphorylation of CD3 ITAMs. Because of its tandem SH2 domains, ZAP-70 then binds to these motifs (55, 68). Recruitment of ZAP-70 to the ITAMs is required for activation, since agents that block recruitment prevent these events (56, 69). The involvement of ZAP-70, along with Lck, in signaling through CD38 is supported by the observation that ZAP-70 becomes tyrosine phosphorylated upon CD38 ligation. In contrast, we failed to detect Syk tyrosine phosphorylation upon CD38 triggering, although both ZAP-70 and Syk belong to the same PTK family, and they are readily tyrosine phosphorylated upon CD3 ligation (Fig. 8). Moreover, CD38 ligation induces a

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Acknowledgments We gratefully acknowledge Isabel de Aos and Dr. Dolores Collado for helpful discussions. We thank Drs. Balbino Alarcbn, Steven C. Ley, and Arthur Weiss for kindly supplying Abs, and Pilar Ruiz for technical assistance.

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MAP KINASE A N D ZAP-70 ACTIVATION BY CD38 LIGATION


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