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(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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