(CANCER RESEARCH 55. 4157-4161. September 15. 1W5|

CD30 Ligand Signal Transduction Involves Activation of a Tyrosine Kinase and ofMitogen-activated Protein Kinase in a Hodgkin's Lymphoma Cell Line1

Clemens-Martin Wendtner, Barbara Schmitt, Hans-Jürgen Gruss, Brian J. Druker, Bertold Emmerich,Raymond G. Goodwin, and Michael Hallek2

LudwiX'Maxitnilians-Univeraitat. Klinikum Innenstadt. Medizinische Klinik, Ziemssenstraße l, D-8Ü336Munich, Germany {C-M. W., B. S.. B. E., M. H.]: Oregon Health Science*University, Portland, Oregon 97201-3098 ¡B.J. D.¡;unii Immune* Research und Development Corporation. Seattle, Washington 98101 [H-J. G., R. G. G.l

ABSTRACT

CD30 is a transmembrane receptor of the nerve growth factor/tumornecrosis factor receptor superfamily. Its expression is associated withHodgkin's lymphoma and a subset of non-Hodgkin's lymphoma. Re

cently, its ligand (CD30L) has been cloned. CD30L enhances the proliferation of peripheral T cells and the Hodgkin's cell line HDLM-2 but

seems to exert antiproliferative effects on large cell anaplastic lymphomacell lines. Since tyrosine kinases are critical regulators of cell growth, weinvestigated whether CD30L induced changes in cellular tyrosine phos-phorylation in CD30-positive lymphoma cell lines. Stimulation with

CD30L or with an agonistic mAb against CD30, M44, induced a rapid,transient, and concentration-dependent tyrosine phosphorylation of acytosolic protein of 17, 42,000 (p42) in the Hodgkin's lymphoma cell line

HDLM-2 but not in other CD30-positive lymphomas. In HDLM-2 cells,the phorbol ester phorbol 12-myristate 13-acetate also stimulated tyrosine

phosphorylation of p42, and this effect was enhanced by M44. In markedcontrast, agents stimulating the protein kinase A pathway, like forskolinor dibutyryl cAMP, did not affect tyrosine phosphorylation of p42. Byimmunoprecipitation with mAbs against mitogen-activated protein kinase(MARK; p42KRK"), a Mr 42,000 protein was identified which comigrated

with p42 on SDS gels and which was phosphorylated on tyrosine residuesin response to stimulation of CD30. Immune complex kinase assaysshowed that M44 mAb induced the activation of MAPK (p42KKK") and

the phosphorylation of a MAPK substrate, niyelin basic protein. Takentogether, the results suggest that CD30L induces the tyrosine phosphorylation and activation of the MAPK p42KKK" isoform in HDLM-2 cells.

These findings may have implications for the understanding of the pallio-genesis of Hodgkin's disease.

INTRODUCTION

CD3U antigen is a marker for HL3 that is expressed on Hodgkin's

and Reed-Sternberg cells as detected by the mAb Ki-1 ( 1). In addition,CD30 expression defines an entity of NHL, the CD3()-positive LCAL

(2). Finally, CD30 is also detected on carcinomas, malignant melanomas, and mesenchymal tumors (3, 4). Besides tumor-associated

expression, CD30 surface molecules are found on activated or virallytransformed T and B cells, as well as on natural killer cell clones andend-stage macrophages (5, 6). The CD30 antigen represents a M,120,000 membrane-bound phosphorylated glycoprotein encoded by a

cDNA that shows sequence homology in its extracellular domain tothe TNF/NGF receptor superfamily (7, 8).

Cloning of the CD30L showed that its cDNA encodes a membrane-

bound protein with homology to the TNF ligand superfamily (9, 10).CD30L is expressed mainly on activated T cells, granulocytes, andmacrophages and exerts pleiotropic effects on distinct CD30-positive

Received 3/7/95; accepted 7/14/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked atlvenisemtni in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' Supported in part by Grant Ha 1680/2-1 from the Deutsche Forschungsgemeinschaft.2 To whom requests for reprints should be addressed.'The abbreviations used are: HL. Hodgkin's lymphoma: NHL. non-Hodgkin's lym

phoma; LCAL. large cell anaplastic lymphoma; TNF. tumor necrosis factor: NGF. nervegrowth factor; CD30L, CD31I ligand; MAPK, mitogen-activated protein kinase; PMA,phorbol 12-myristate 13-acetate; ram, rabbit antimousc IgGl: PKC. protein kinase C;PKA, protein kinase A; MBP. myelin basic protein.

lymphoma subtypes (9, 11). Recombinant human CD30L as well asthe agonistic anti-CD30 mAb, M44, directed against the extracellular

portion of the CD30 antigen, were both shown to support the proliferation of HL cell lines with T-cell-like phenotype (e.g.. HDLM-2 andL-540), whereas there is no proliferative effect in HL cell lines withB-cell-like type (e.g.. KM-H2 and L-428; Ref. 11). In contrast, thegrowth of CD30-positive LCAL cell lines, such as Karpas-299, was

inhibited by CD30L (11). It was hypothesized that the pleiotropiceffects of CD30L are due to different intracellular signaling pathwaysor a consequence of CD30 cDNA variants among the different lymphoma cell lines (12).

It is well established that tyrosine kinases and phosphatases arecritical regulators of cell growth. Several protein (tyrosine) kinasecascades that are activated by cytokines and growth factors have beenidentified during the past 10 years (13-15). In particular, a growthfactor-dependent pathway now referred to as the Ras signaling path

way has been characterized that involves the coordinate activation oftwo proto-oncogene products, p21ras and Raf-1, which subsequently

activate MAPK (16, 17). Various cytokines and growth factors, including NGF and TNF, have been reported to activate the Ras signaling pathway and MAPK (18-22). Little is known about the sig

naling events initiated by the interaction of CD30L with its receptor.The recent cloning of both CD30L and CD30 has provided the toolsto study the transmembrane signaling induced by CD30L/CD30 coupling. In the present study, we investigated whether stimulation withCD30L is associated with changes in tyrosine phosphorylation ofcytosolic proteins in different CD30-positive lymphoma cell lines. We

could demonstrate that one cytosolic protein, p42, was phosphorylatedin response to CD30L in a HL cell line, HDLM-2. Additional exper

iments with monospecific antibodies against MAPK allowed us toidentify this protein as one isoform of MAPK.

MATERIALS AND METHODS

Reagents. M44, an agonistic mAb (murine IgGl) against human CD30,as well as CV-1/EBNA cells transfected with the human CD30L cDNAcontaining expression vector (CV-1/CD30L) or the expression vector alone(CV-1/mock), respectively, were generated as described previously (9). BSA

(fraction V), PMA, forskolin, and dibutyryl cAMP were purchased from SigmaChemical Co. (Munich. Germany), ram (Fc-y fragment-specific) was used for

cross-linking and was obtained from Dianova (Hamburg. Germany). All re

agents for cell lysis, protein extraction, SDS-PAGE, and immunoblotling werepurchased from Sigma or Bio-Rad (Munich, Germany). The anti-phospnoty-rosine mAb 4G1Ãœis specific for tyrosine-phosphorylated proteins and does notcross-react with phosphoserine, phosphothreonine. phosphohistidine, or tyrosine sulfate.4 Rabbit polyclonal anti-Nek antibody was kindly provided by Dr.

U. Lammers (Max-Planck-Institut fürBiochemie, Martinsried, Germany). An-ti-MAPK (ERKI, ERKII, ERKI-III, and ERKCT) and anti-She mAbs werepurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [•y-12P]ATP

was obtained from Amersham (Braunschweig, Germany).Cells and Culture. The human cell lines HDLM-2. KM-H2, and Karpas-

299 were obtained from the German Collection of Microorganisms andCell Cultures (DSM. Braunschweig, Germany). HDLM-2 was originally

4 B. Druker. unpublished dula.

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CTOO I.KÃŒANDSIGNAL TRANSACTION

established from the pleural effusion of a 74-year-old male with Hodgkin's

disease (nodular sclcrosing, stage IV; Ref. 23). HDLM-2 cells were cul

tured in RPMI Ki4() (GIBCO, (ìaithersburg. MD) supplemented with 20%FCS. 1(10fig/ml penicillin. 100 fig/ml streptomycin, and 2 mM i.-glutamineat 37°Cin a humidified. 5% CO, atmosphere. The Karpas-2lW cell line was

established from blast cells in the peripheral blood of a 25-year-old male

with LCAL (24) and was maintained in RPMI 164(1 supplemented with10% PC'S. The same culture conditions were used for KM-H2. a human cell

line established from the pleural effusion of a 37-year-old male withHodgkin's disease (mixed cellularity, stage IV; Ref. 25).

SI iuni lut iini ni Cells and Analysis of Tyrosine Phosphorylation. Prior tostimulation, exponentially growing cells were serum deprived by incubation inRPMI l()4(l supplemented with \% BSA for 16 h. In standard experiments.5 X 10" cells were stimulated with 2 x IO5 CV-1/CD30L or CV-1/mock. or

with 10 fig M44 mAb in a total volume of 1 ml at 37°Cfor 5 min. For the

investigation of the effects of PKC and PKA pathways, cells were stimulatedwith CD30L or M44 after incubation with PMA (0.1-1 JJ.M). forskolin. ordihutyrvl cAMP (both 0.1-1 mM). After stimulation, cells were washed in cold

PBS. lysed in KM)pii NP40 buffer |20 mM Tris base (pH 8.0). 137 mM NaCI.10% glycerol. and \7t NP40| containing inhibitors [1 mM phenyl methyl-

sulfonyl fluoride (Sigma), 0.15 unit/ml aprotinin (Sigma). IO min EDTA.10 /xg/ml leupeptin (Sigma). 100 mM sodium fluoride, and 2 /XM sodiumorthovanadate] at 4°C for 20 min. Insoluble materials were removed bycentrifugaron at 10.000 X ¡>for 15 min at 4°C.The supernatant was removedand frozen at -20°C until used.

Lysates of 5 X 10" cells, typically yielding about 200 /¿gprotein as

determined by the Bio-Rad protein assay, were mixed at a 1:1 ratio withsample buffer containing \(}ri (w/v) SDS and 28 mM 2-mercaptoethanol andheated at 100°Cfor 5 min before loading the samples on SDS gels. Proteins

were separated by 7.5% SDS-PAGE, transferred onto Immohilon P transfer

membranes (Millipore Corporation. Bedford, MA), and immunohlolted withthe anti-phospholyrosine mAh 4GIO as described previously (26. 27). Briefly,

filters were blocked by incubation in TBS [10 mM Tris (pH 8.0) and 150 mMNad| with 5% BSA for l h at 25°C immediately after the transfer and

incubated overnight with the primary antibody (4GIO; diluted 1:2500 in TBS).Membranes were washed in TBS followed by incubation with the secondaryantibody, an alkaline phosphatase-conjugated anti-mouse IgG (Bio-Rad: di

luted 1:5000 in TBS). Finally, membranes were washed in TBS and developedwith nitro blue lelrazolium and 5-bromo-o-4-chloro-3 indolvlphosphatc (Bio-

Rad).lmimiini|nv< ipilalion. ImmiinoprecipiUitions were performed using poly-

clonal antibodies against Nek. She. and MAPK (ERKI. ERKII. ERKI-III. andERKCT) and protein A-Sepharose beads (Pharmacia. Freiburg. Germany).Protein A heads were washed with lysis buffer, and NXI-jnl aliquots of the

slurry were then mixed with cell lysates for preclearing. Thereafter, theantibody (5 fig) was added, and the reaction mixture was incubated for 4 h at4°Con a rotator. Precipitates were washed several times in lysis buffer and

boiled for 5 min; then proteins were separated by 10% SDS-PAGE.Immune Complex Kinase Assays with MAPK Substrate MBP. Immu-

noprecipitations with MAPK mAbs were performed as described above: inaddition, precipitates were subsequently incubated for 15 min in kinase buffer[5(1 mM Tris (pH 7.4) and 10 mM MnCUj supplemented with 10 /¿Ci[•y-'2P|ATPand, in some experiments, with 40 /^g MBP/reaction. The reaction

was terminated by adding 2x sample buffer and boiling lor 5 min. Proteinswere resolved by 15%. SDS-PAGE and visualized by autoradiography.

RESULTS

CD30L Induces Tyrosine Phosphorylation of a Mr 42,000 Protein in the Hodgkin's Cell Line HDLM-2. To explore the differen

tial effects of CD30L on tyrosine kinase activity of CD3()-positive HLand NHL cells, the tyrosine phosphorylation of cytosolic phospho-

proteins in response to CD30L was investigated in the cell linesHDLM-2, KM-H2, and Karpas-299. Cells were serum deprived for 16h and then stimulated for 5 min with CD30L or the CD3()-activating

mAh M44. After lysis, proteins of the soluhle fraction were suhjectedto 7.5% SDS-PAGE, followed by immunohlotting with the anti-phosphotyrosine mAh 4O10. As shown in Fig. 1, both CD30 cross-

KM-H2 HDLM-2

/ HIt S* t*

=••••»»•kPa

205 — —

116.5-

80 -

49.5-

ü O

p42

32.5 -

Fig. I. Effect of recomhinant human CD3IIL and anli-C'D30 mAh M44 on tyrosinephusphorylatinn of proteins in HL cell lines. HDLM-2 and KM-H2 cells (both 5 X IO6/

ml) were stimulated with medium (control), rimi (II) ng/ml). or ram und M44 (both H)fig/ml). In addition. HDLM-2 cells (5 X 10"/ml) were incubated with CVl/mock orCV1/CD30L cells (both 2 X llf/ml). Lysates were resolved on 7.5r'r SDS-PAGE and

immunohlotted with anliphosphotyrosinc mAh 4(ilO. A protein of about M, 42.IMKÕ(p42)was detected upon specific stimulation (tirrow).

linking (with M44 antibody and ram) and stimulation withCV-1/CD30L increased the tyrosine phosphorylation of a protein with

an apparent molecular weight of 42.000 (p42) in the HL cell lineHDLM-2. In cells stimulated with ram or CVl/mock, tyrosine phos

phorylation of p42 was not increased as compared to controls stimulated with media alone (Fig. 1). In marked contrast, no apparentincrease in the tyrosine phosphorylation of cytosolic proteins wasdetectable in the HL cell line KM-H2 in response to CD30 stimulation(Fig. 1). Similar results were obtained for the NHL cell line Karpas-

299 (data not shown).Subsequent experiments using different concentrations of M44

mAb showed that its effect on HDLM-2 cells was dose dependent,

with a maximal tyrosine phosphorylation of p42 observed at concentrations as low as 1 ju.g/ml M44 mAb (Fig. 2A). Time course experiments with the HDLM-2 cells demonstrated that CD30 stimulation

by M44 mAb (10 /j,g/ml) induced a rapid and transient tyrosinephosphorylation of p42 (Fig. 2B). Tyrosine phosphorylation of p42was seen as early as 1 min after CD30 stimulation, peaked at 10 min.and decreased thereafter.

Both CD30 Receptor Activation and PMA Induce TyrosinePhosphorylation of p42. After having established that at least onecellular protein was activated by CD30L, we asked whether othersignal transduction intermediates would interfere with tyrosine phosphorylation of p42. Tyrosine phosphorylation of cellular proteins canbe modulated by PKC activators such as PMA (2X). Therefore, weasked whether PMA had any effect on the phosphorylation status ofp42. When HDLM-2 cells were stimulated by PMA at optimal con

centrations (l JU.M),a protein comigrating with p42 on SDS gelsbecame tyrosine phosphorylated (Fig. 3). Preincubation of cells withPMA at suboptimal concentrations (0.1 /J.M)did not stimulate tyrosine

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(TOI! IJ(iANI) SIGNAI. TKANSIHKTION

AkDa

205

116.5

80

10 5 3 1 0.1 O

mmmmmm

M44)

49.5 -|

32.5 -

B

kDa

49.5

p42

10" 1' 5' 10' 30' 60' 8

p42

32.5 --

with different anti-MAPK niAbs, separated by SDS-PAGE, and im-munoblotted with the anti-phosphotyrosine antibody 4GK). As shown

in Fig. 4, stimulation with M44 niAb induced a subtle but reproducible increase in tyrosine phosphorylation of a M, 42,000 protein thatcould be purified by an anti-ERKII antibody and comigrated with p42on SDS gels. MAPK antibodies with specificity for ERRI. ERKI-III,or ERKCT did not allow precipitation of MAPK in M44 mAb-stimulated HDLM-2 cells (data not shown). Based on these experiments in which p42 was recognized only by the anti-ERKII MAPKantibody, p42 was likely to be identical with p42MAI'K (p42l:RK").

Other candidates for p42 were Nek, a M, 47,000 protein kinasewhich shows enhanced phosphorylation in response to stimulation ofthe NGF receptor in PC 12 cells as well as after treatment with PMAin A431 cells (31), and She, a phosphoprotein known to activate Ras,which exists in isoforms of M, 46,000 and M, 52,000 (32). However,we were not able to demonstrate tyrosine phosphorylation of Nek orShe proteins in response to CD30 activation (data not shown).

To further substantiate the hypothesis of p42 being a MAPKisoform, we investigated whether p42' kk" kinase was activated upon

CD30 stimulation. Lysates of unstimulated and M44-stimulatedHDLM-2 cells were immunoprecipitated with the anti-ERKII anti

body, and immune complex kinase assays were performed by adding[y-32P]ATP to the reaction. Upon CD30 stimulation by cross-linking

with M44 mAb, the phosphorylation of a A/r 42,000 protein precipitated by the anti-ERKII antibody was strongly increased as compared

to stimulation with media or ram, suggesting a specific phosphorylation of p42HRK" in response to CD30 stimulation (Fig. 5A). Toassess the activity of p42'"RK" in response to CD30 activation, weinvestigated next whether p42r RK" could phosphorylate the MAPK-

spccific substrate, MBP (33). MBP (40 ng/ml) was added to the

M44:

Fig. 2. Tyrosine phosphoryUition of p42 in response lo CD30 cross-linking: concentration and time kinetics. HDLM-2 cells were stimulated hy M44/CD30 cross-linking andlysed; then cylosolic proteins were extracted and separated hy 7.5% SDS-PAGE and

immunohlotted wilh 4G10 after specific stimulation with M44 mAh at various concentrations (A) or at various time points (H) as indicated.

PMA:(MM)

0.1 1.0

\ I

phosphorylation of p42. However, when M44 mAb (10 fig/ml) wasadded subsequently, an increase in tyrosine phosphorylation of a M,42,000 protein was detected. In summary, these experiments demonstrate that M44 mAb induces the tyrosine phosphorylation of p42 inan additive manner with PMA. We also investigated whether thesecond messenger cAMP interferes with phosphorylation of p42 byM44 mAb. HDLM-2 cells were prestimulated for 5 min with thecAMP analogue dibutyryl cAMP (0.1-1 mM) or with forskolin (0.1-1

ITIM).In marked contrast to the effects of PMA and M44 mAb,dibutyryl cAMP or forskolin did not change the tyrosine phosporyla-tion of p42 in unstimulated or M44-stimulated HDLM-2 cells (data

not shown).Stimulation of CD30 Induces Phosphorylation of MAPK and of

Its Substrate MBP. In an effort to identify p42, mAbs against knowntyrosine phosphoproteins with a molecular weight of about 42,000were used in immunoprecipitation experiments. PMA and TNF wereboth shown to activate MAPK (24). In our experiments, both PMAand CD30L induced the tyrosine phosporylation of a protein of Mr42,000, which is the molecular weight of at least one MAPK isoform(18, 30). Given this coincidence, the MAPK p42liRK" became a

primary candidate to be investigated. Therefore, MAPK protein complexes were purified from whole-cell lysates by immunoprecipitation

4159

106 _

80 -

49.5-

32.5 -

27.5 -

Fig. 3. Effect of PMA on tyrosine phosphorvlation of p42. HDLM-2 cells wereprcstimulaled with PMA at concentrations of 0.1-1 ¿IMfor 5 min. and M44 niAb wasauded where indicated ( + ). Cells were lysed, separated by 7.5% SDS-PAGE, andimmunoblotied with 4G10.

p42

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CD30 LIOAND SIGNAL TRANSDUCTION

Native a-erkll IP

P42

Fig. 4. Immunoprecipitation with anti-ERKII MAPK antibody. HDLM-2 cells werestimulated with medium alone ( —)or ram and M44 mAb ( + ). Cells were lysed, andwhole-cell lysates were analyzed by direct immunoblotting with mAb 4G10 (Native} orprecipitated with anti-ERKII MAPK antibody before 4G10 immunoblotting (a-ERKllIP). Lysates were resolved on 10% SDS-PAGE.

kinase reaction after immunoprecipitation of CD30-stimulated andunstimulated HDLM-2 lysates with the anti-ERKII antibody.Although the increase of p42ERK" phosphorylation in response to

CD30 stimulation was less prominent than in the previous experiment,the phosphorylation of the MAPK substrate MBP clearly increased(Fig. 5B). Taken together, the experiments strongly suggest that a M,42,000 protein, which is precipitated by a monoclonal anti-ERKIIantibody, is activated in response to CD30 stimulation in HDLM-2cells.

to observations in the human myeloid cell line MO7, where PMA wasshown to augment the granulocyte-macrophage colony-stimulatingfactor-induced tyrosine phosphorylation of a MT42,000 phosphopro-tein later identified as p42MAPK (28, 38). In marked contrast, com

pounds known to stimulate PKA apparently did not affect p42 tyrosine phosphorylation. However, our data do not rule out the possibilitythat the cellular effects of CD30L on HL cells are fine-tuned by asignaling cross-talk between PKA and the Ras—»Raf-1 —»MAPK

pathway, as shown in fibroblasts or PC12 cells (39).With regard to the biological effects of CD30L, pleiotropic effects

on different CD30-positive lymphoma subtypes have been described.In HL cell lines with T-cell-like phenotype, CD30L induced cell

proliferation; in contrast, CD30L had no effect on the growth of HLcell lines with B-cell-like phenotype and antiproliferative effects with

cytolytic cell death in a subgroup of NHL cell lines derived fromCD30-positive LCAL (11). Therefore, a cytokine receptor function

for the CD30 antigen with CD30L as the specific activating cytokinewas postulated. Our results suggest that some of the pleiotropic effectsof CD30L in these different lymphoma entities might be caused bydifferential effects on intracellular signaling pathways. Along theselines, MAPK was only activated in the T-cell-like HDLM-2 cell line,

which showed a proliferative response to CD30L. However, it remains to be determined whether MAPK activation is essential for themalignant growth of Hodgkin's and Reed-Sternberg cells and for the

proliferative effects of CD30L. The recent finding that constitutivelyactive MAPK kinase has transforming activity for mammalian cells(40) justifies the speculation that a (constitutive) activation of MAPKby CD30L/CD30 coupling might promote the growth of Hodgkin's

lymphoma or other human CD30-positive tumors. However, furtherinvestigations, including studies with dominant-negative mutants of

ERKII and antisense oligodesoxynucleotides, are needed in order to

DISCUSSION

MAPKs are members of highly conserved pathways in eukaryoticcells from yeast to humans and are involved in the regulation ofdiverse cellular functions like osmoregulation, cell wall biosynthesis,cell growth, and differentiation (34). It was shown that ligand-medi-

ated stimulation of both the NGF receptor and the TNF receptoractivates MAPKs by tyrosine phosphorylation (21, 22, 33, 35). Ourstudy shows that activation of CD30, another member of this cytokinereceptor superfamily, increases the phosphorylation and activity of aMAPK, p42ERK" in a Hodgkin's cell line. Although our data do not

formally exclude the possibility that additional Mr 42,000 phospho-

proteins are also involved in this pathway, they strongly suggest thatp42ERK" is at least one signaling intermediate activated in response to

CD30L/CD30 coupling. The pathway by which CD30L/CD30 coupling activates MAPK is unknown. Activation of the Ras signalingpathway, which mediates its effects through a kinase cascade involving the serine-threonine kinase Raf-1 and the tyrosine/threonine

kinase MAPK kinase, might be a possibility (36). It has been shownthat TNF-a uses this pathway. PKC is known to activate theRas—»Raf-1—»MAPK pathway as well (37), consistent with our find

ing that the phorbol ester PMA, which activates PKC, also induced thetyrosine phosphorylation of p42 (Fig. 3). The additive modulation ofp42 (MAPK) tyrosine phosphorylation by CD30L and PMA is similar

B

p42

p42

MBP

Fig. 5. Stimulation of CD30 induces phosphorylation of MAPK and its substrate MBP.HDLM-2 cells were stimulated with medium (control), ram. or ram and M44 mAb.lysed, and precipitated by anti-ERKII MAPK antibody. A, [-y-MP]ATP was added to the

precipitates, and an immune complex kinase reaction was performed as described in"Materials and Methods." ß,the MAPK substrate MBP (M, ~ 18,000) was added to the

immune complex kinase reaction, and its phosphorylation status was evaluated by 15%SDS gel electrophoresis and subsequent autoradiography for 48 h.

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(TOI! I.IGANI) SIGNAI. IRANSDUCTION

establish a role of MARK activation by CD30/CD30L coupling for thepathogenesis of Hodgkin's lymphoma.

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1995;55:4157-4161. Cancer Res Clemens-Martin Wendtner, Barbara Schmitt, Hans-Jürgen Gruss, et al. Hodgkin's Lymphoma Cell LineTyrosine Kinase and of Mitogen-activated Protein Kinase in a CD30 Ligand Signal Transduction Involves Activation of a

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