Full text of DOI:10.1080/15216540050212132

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IUBMB Life, 50: 119 124, 2000
c
°
Copyright 2000 IUBMB
1521-6543/00 $12.00 + .00
Original Research Article
Sphingosine 1-Phosphate Activates Erk-1/-2
by Transactivating Epidermal Growth Factor
Receptor in Rat-2 Cells
Joo-Hee Kim, Jung-Hee Kim, Woo Keun Song, Jae-Hong Kim, and Jang-Soo Chun
Department of Life Science, Kwangju Institute of Science and Technology, Pook-Gu, Kwangju,
500-712, Korea
nously added S1P binds to prototypical members of the orphan
(
)
endothelial differentiation gene EDG family, which is linked to
Summary
We investigated a signaling pathway leading to activation of ex-
( )
Gi protein in many cell types 4 . When stimulatedby S1P, EDG
–
(
)
tracellular signal regulated protein kinase Erk 1 and 2 in Rat-2
receptors modulate a wide spectrum of signaling pathways, in-
cluding extracellular signal regulated protein kinase Erk-1/-2
5 9 , phospholipase C PLC , and consequently Ca mobi-
(
)
cellsstimulatedwithsphingosine 1-phosphate S1P . S1P treatment
transiently activated Erk-1/-2 in a dose-dependent manner, and its
activation was blocked by pertussis toxin, expression of RasN17, or
inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2
was also suppressed by the inhibition of epidermal growth factor
–
(
)
+
2
–
(
)
(
)
–
(
)
lization 10 12 .
Despite extensive studies on Erk-1/-2 activation by S1P in
various cell types, the precise signaling mechanisms leading to
Erk-1/-2 activation remain undetermined. In many cell types,
S1P appears to activate Erk-1/-2 through stimulating the EDG
–
(
)
EGF receptor tyrosine kinase with the speciŽ c inhibitor AG1478,
suggesting that activation of EGF receptor tyrosine kinase was in-
volved in the signaling pathway. S1P-induced Erk-1/-2 activation
(
)
was enhanced up to 2-fold by inhibiting protein kinase C PKC
(
)
family of receptors by way of pertussis toxin PTX sensitive
with GF109203X, and PKC inhibition in the absence of S1P treat-
ment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2
activation by PKC inhibition was blocked by treating cells with
AG1478, suggesting the involvement of PKC in the regulation of
EGF receptor tyrosine kinase activation that leads to Erk-1/-2 ac-
tivation. Together, these results suggest that S1P activates the EGF
receptor through a PKC-dependent pathway that links Ras signal-
ing to the activation of Erk-1/-2 in Rat-2 cells.
G proteins by S1P treatment is coupled to various signaling
( )
molecules, including tyrosine kinase c-Src 8 , phosphatidy-
linositol 3-kinase 8 , PLC, and protein kinase C PKC 6 ,
leading to an activation of Erk-1/-2.
Because stimulation of PTX-sensitive G proteins with cer-
tain agonists is known to activate Erk-1/-2 by transactivating the
( )
(
) ( )
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IUBMB
Life, 50: 119 124, 2000
(
)
epidermal growth factor EGF receptor, which functions as a
(
)
scaffold for the recruitment of signaling proteins 13, 14 , we
examined a role of EGF receptor transactivation in S1P-induced
Erk-1/-2 activation to provide further insight into the mecha-
nisms underlying the S1P actions in Erk-1/-2 action. Here, we
report studies with Rat-2 cells in which S1P activates Erk-1/-2
in a PTX-sensitive manner through transactivation of the EGF
receptor as mediated by inhibition of PKC. We found that S1P-
Keywords EGF receptor; Erk-1/-2; protein kinase C; sphingosine
1-phosphate.
INTRODUCTION
1
(
)
Sphingosine 1-phosphate S1P , one of the platelet-derived
lipid mediators, regulates various diverse cellular functions, in-
cluding proliferation, apoptosis, neurite retraction, tumor inva-
–
–
induced EGF receptor transactivationis cell type speciŽ c, being
observed in Rat-2 cells but not in HeLa, NIH3T3, or SK-N-
(
)
siveness, metabolism, and morphological changes 1 3 . Al-
though whether S1P actsintracellularlyasasecond messenger or
extracellularly as a receptor ligand, or both, is unknown, exoge-
( )
BE2 C cells.
1
(
)
Abbreviations: AEBSF, 4- 2-aminoethyl benzenesulfonyl  uoride; EDG,
endothelial differentiation gene; EGF, epidermal growth factor; Erk, extracellu-
Received 6 July 2000; accepted 25 August 2000.
Address correspondence to Dr. Jang-Soo Chun. Fax: 82-62-970-
2484; E-mail: jschun@eunhasu.kjist.ac.kr
–
lar signal regulated protein kinase; PAGE, polyacrylamide gel electrophoresis;
PLC, phospholipase C; PKC, protein kinase C; PTX, pertussis toxin; SDS,
sodium dodecyl sulfate; S1P, sphingosine 1-phosphate.
119

120
KIM ET AL.
EXPERIMENTAL PROCEDURES
Expression of RasN17 was conŽ rmed by Western blot analysis
with an antibody that recognizes both Ras and RasN17 proteins.
Immuno uorescence microscopy revealed that >90% of cells
( )
Cell Culture. Rat-2, NIH3T3, and SK-N-BE2 C cellswere
grown in Dulbecco’s modiŽ ed Eagle’s medium supplemented
with 10% v/v heat-inactivated bovine calf serum, 50 l g/ml
(
)
expressed a substantial amount of RasN17 data not shown .
(
)
(
streptomycin, and 50 units/ml penicillin Gibco-BRL, Gaithe±rs-
RESULTS
)
(
)
burg, MD in a humidiŽ ed atmosphere of air/CO2 19:1 at 37 C
(
)
as described previously 15 . HeLa cells were maintained in
Exogenous S1P Activates Erk-1/-2 in a PTX-Sensitive Man-
(
)
RPMI 1640 medium supplemented with 10% v/v bovine calf ner inRat-2 Cells. Erk-1/-2 activationwas determinedbyWest-
(
)
serum and antibiotics as described previously 15 .
ern blot analysis with antibody that recognized the activated,
Erk-1/-2 Assay. Activation of Erk-1/-2 in stimulated cells tyrosine- and threonine-phosphorylated form of the enzyme.
was determined by using antibody speciŽ c to activated Erk-1/-2 Fig. 1 shows dose-dependent activation of both Erk-1 and Erk-2
(
)
as described previously 16, 17 . Brie y, after cells were serum- and the time course of activation in S1P-treated Rat-2 cells. S1P
starved for 6 h, proteins were extracted with a buffer containing activated the enzyme transiently; its activity peaked at 10 min
(
)
50 mM Tris-HCl,pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% after S1P treatment and declined thereafter Fig. 1B . Phospho-
SDS, and various inhibitors of proteases—10 l g/ml leupeptin, rylation of Erk-1/-2 was detected when as little as 0.01 lM S1P
(
)
(
)
10 lg/mlpepstatinA, and 1 mM 4- 2-aminoethyl benzenesulfo- was used, the maximum effect being obtained at 1 lM Fig. 1A .
(
)
nyl  uoride AEBSF —and of phosphatases—5 mM sodium We therefore used 1 lM S1P in the subsequent experiments.
(
)
 uoride, 1 mM sodium orthovanadate. Proteins 30 lg were S1P-induced Erk-1/-2 activation was suppressed by pretreating
–
(
)
(
)
size-fractionated by sodium docecyl sulfate SDS 8% poly- cells with 100 ng/ml PTX for 24 h Fig. 1B , which suggests
(
)
acrylamide gel electrophoresis PAGE and transferred to a ni- the involvement of PTX-sensitive G proteins in the S1P action.
trocellulose membrane. Phosphorylated Erk-1/-2 was detected Although we did not directly prove ADP-ribosylation of Gi/Go
by Western blotting with antibody speciŽ c to Erk-1/-2 that had proteins by PTX, the results are consistent with previously re-
(
beenactivatedandtyrosine-andthreonine-phosphorylated New ported observations in many other cell types that S1P activates
–
)
England Biolabs, Beverly, MA .
Erk-1/-2 by way of PTX-sensitive G protein coupled receptors
(
)
Raf Kinase Assay. Cells, either untreated or treated with
6, 9, 10 .
various reagents as indicated in each experiment, were extracted
S1P Activation of Erk-1/-2 Is Ras, Raf, and MEK Dependent.
with 50 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 1% Activation of Erk-1/-2 in Rat-2 cells stimulated with 1 lM S1P
Triton X-100, 2 mM EDTA, 1 mM EGTA, 10 l g/ml leupeptin, was blocked when the cells were pretreated with PD98059, a
10 l g/ml pepstatin A, and 1 mM AEBSF. A 500-lg portion speciŽ c inhibitor of MEK-1/-2 that activates Erk-1/-2 by dual
–
(
)
of proteins was incubated with 1 lg of anti c-Raf-1 polyclo±nal phosphorylation 19 . A role for c-Raf-1, an intermediate be-
(
)
antibody Santa Cruz Biotech, Santa Cruz, CA for 2 h at 4 C, tween Ras and MEK, in Erk-1/-2 activation was examined by
and the immune complex was incubated with protein A-coated treating the cells with forskolin. Forskolin is known to induce
±
(
)
beads Pierce, Rockford, IL for 1 h at 4 C. The precipitated inhibitory phosphorylation of c-Raf-1 by increasing cAMP and
immune complex was incubated for 30 min at 30 ±C with 0.5 consequently activating protein kinase A 20, 21 . Treating the
(
)
(
lg of unphosphorylated MEK-1 Upstate Biotechnology, Lake cells with forskolin completely inhibited S1P-induced Erk-1/-2
32
)
(
)
[
]
Placid, NY as a Raf substrate and 1 lCi of c - P ATP in 30 ll activation Fig. 2A . Raf kinase activity, as determined by the
of the reaction buffer: 25 mM Tris, pH 7.4, 10 mM MgCl2, 0.5 immune complex kinase assay, was transiently increased by S1P
(
)
mM EGTA, 10 lg/ml leupeptin, 10 l g/ml pepstatin A, 1 mM treatment but was blocked by forskolin treatment Fig. 2C . The
AEBSF, 5 mM NaF, 1 mM sodium orthovanadate, and 1 mM involvement of Ras, an upstream signaling molecule of Raf, was
4-nitrophenyl phosphate. The reaction was stopped by adding conŽ rmed by the observation that expression of the inhibitory
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SDS sample buffer, and proteins were size-fractionated by SDS
RasN17 completely blocked S1P-induced Erk-1/-2 activation
32
(
)
[
]
8% PAGE. Phosphorylation of MEK-1 with c - P ATP was
Fig. 2D . Overexpression of RasN17 did not alter the expres-
(
)
( )
sion pattern of Erk-1/-2 as shown in Fig. 2D lower panel . To-
detected by autoradiography 15 .
Transfection. Functioning of cellular Ras proteins was gether, the above results indicate that S1P activates Erk-1/2 by
blocked by overexpressing anegative mutantof Ras,Ha-RasN17. way of the Ras/Raf/MEK signaling cascade in Rat-2 cells.
Substitution of asparagine for serine at position 17 is known to
greatly decrease the afŽ nity of Ras for GTP, thereby marking sine Kinase in a Cell Type SpeciŽc Manner. Many signaling
S1P Activates Erk-1/-2 by Activating EGF Receptor Tyro-
–
(
)
this as a dominant negative mutant of Ras 18 . Transfection of molecules that stimulate PTX-sensitive G proteins are known to
(
)
the expression vector containing cDNA for the negative mutant activateErk-1/-2 bytransactivatingtheEGF receptor 13, 14, 22 .
(
)
of Ras, RasN17, was performed as described previously 15 . Using EGF as a positive control for stimulation of the EGF re-
(
)
The expression vector 5 l g was introduced into Rat-2 cells by ceptor and Erk-1/-2, we therefore addressed the possibility that
(
)
using LipofectaminePLUS Gibco-BRL as recommended by S1P induced activation of the EGF receptor as an intermediate
the manufacturer. The transfected cells, which were cultured in step in a signal transduction pathway that was linked to Erk-1/-2
complete medium for 48 h, were used for the Erk-1/-2 assay. activation. As shown in Fig. 3A, EGF-induced activation of

121
SPHINGOSINE 1-PHOSPHATE ACTIVATION OF ERK-1/-2
Involvement ofPKCinEGF ReceptorTransactivation. Given
reports that inhibition of PKC unmasked EGF receptor activation
–
(
)
as a result of G protein coupled receptor activation 22, 24 , we
next examined whether PKC participated in S1P-induced Erk-
1/-2 activation. Activation of Erk-1/-2 by S1P treatment was
markedly enhanced when the PKC was inhibited with
(
)
(
)
GF109203X Fig. 5 or Go6976 data not shown . Inhibition
of PKC with GF109203X in the absence of S1P treatment also
caused a slight activationof Erk-1/-2, whereas pretreatment with
AG1478 blocked the GF109203X-induced Erk-1/-2 activation.
Together, the results suggest that inhibition of PKC acts as an
intermediate in transactivation of the EGF receptor.
DISCUSSION
To our knowledge, this is the Ž rst demonstration that S1P-
induced activation of Erk-1/-2 involves activation of EGF re-
ceptor tyrosine kinase in a PTX-sensitive manner in Rat-2 cells.
–
StimulationofcertainG protein coupledreceptors iswell known
to transactivate the EGF receptor, which leads to Erk-1/-2 acti-
vation through recruitment of the adapter proteins Shc and Grb2
–
(
)
25 29 . We think it likely, therefore, that activation of EGF re-
ceptor tyrosine kinase by treatment with S1P also recruits these
adaptor proteins, which couple theactivatedreceptor tothe guan-
(
ine nucleotide exchange protein mSos mammalian homolog of
)
son of sevenless and thereby activate the Ras pathway in Rat-2
cells.
Interestingly, the involvement of EGF receptor tyrosine ki-
–
nase in Erk-1/-2 activation was cell type speciŽ c: It was ob-
( )
served in Rat-2 cellsbut not in HeLa, NIH3T3, or SK-N-BE2 C
Figure 1. S1P activates both Erk-1 and -2 in a PTX-sensitive
cells. Although we did not address the underlying mechanism
for the cell type speciŽ city, it might re ect the diverse nature
of S1P signaling. For instance, S1P-induced Erk-1/-2 activation
was both sensitive to PTX and independent of PTX, depend-
–
( )
manner in Rat-2 cells. A Rat-2 cells were serum-starved for
6 h and stimulated with the indicated concentrations of S1P for
( )
10 min. B The cells were treated without or with 100 ng/ml
PTX for 24 h and stimulated with 1 l M S1P for the indicated
(
)
ing on the cell type examined 6, 8 10 . In C6 glioma cells,
S1P-induced activation of Erk-1/-2 was mediated by at least two
signaling pathways—the PTX-sensitive Gi/Go protein pathway,
(
)
(
)
times. Activation of Erk-1 upper band and Erk-2 lower band
was determined by examining phosphorylation of the enzymes
by Western blot analysis. The amount of Erk-2 present was de-
termined by Western blot analysis as a loading control.
( )
and the toxin-insensitive Gq/G11-PLC-PKC pathway 6 . On the
other hand, Erk-1/-2 inairway smooth muscle cellswas activated
after S1P treatment by signaling pathways that are dependent on
( )
Erk-1/-2 in Rat-2 cells was, as expected, blocked by treatment c-Src, Grb-2, and phosphatidylinositol 3-kinase 8 .
–
(
)
with AG1478, a speciŽ c inhibitor of EGF receptor kinase 23 .
The mechanism linking the activated G protein coupled re-
Pretreating the cells with AG1478 also almost completely
ceptors to the EGF receptor was unclear. However, PKC ap-
peared to act as an intermediate of the EGF receptor transacti-
vation in Rat-2 cells, because inhibition of PKC potentiated the
(
)
blocked S1P-induced Erk-1/-2 activation Fig. 3A , thus sug-
gesting the involvement of EGF receptor activation in Erk-1/-2
signaling. The inhibitory effects of AG1478 on EGF- and S1P- S1P-induced Erk-1/-2 activation that was blocked by inhibition
(
)
induced Erk-1/-2 activation was dose-dependent Fig. 3B . In
contrast to Rat-2 cells, however, inhibition of EGF receptor ac-
tivation by AG1478 did not affect S1P-induced Erk-1/-2 activa-
of the EGF receptor tyrosine kinase. Interestingly, inhibition of
PKC with GF109203X in the absence of S1P treatment also
activated Erk-1/-2, and pretreatment with AG1478 blocked the
GF109203X-induced Erk-1/-2 activation. Therefore, we specu-
( )
tion in HeLa, NIH3T3, and SK-N-BE2 C cells, whereas EGF-
induced Erk-1/-2 activation was effectively blocked by AG1478 late that in the absence of S1P treatment, the basal activity of
(
)
in these cells Fig. 4 . Therefore, the activity of EGF receptor
PKC blocks EGF receptor tyrosine kinase activity, which masks
activation of the EGF receptor tyrosine kinase, whereas inhibi-
tyrosine kinase as an intermediate in Erk-1/-2 activation appears
–
–
to be cell type speciŽ c.
tion of PKC, as a result of G protein coupled receptor activation,

122
KIM ET AL.
( )
Figure 2. S1P activates Erk-1 and -2 by way of a Ras-, Raf-, and MEK-dependent signaling pathway. A Serum-starved Rat-
2 cells were pretreated with vehicle alone, 10 l M PD98059, or 50 l M forskolin for 30 min and then stimulated with 1 l M
S1P for the times indicated. Alternatively, the cells were transfected with 5 l g of RasN17 expression vector, cultured for 48 h,
( )
and stimulated with S1P. Dual phosphorylation of Erk-1/-2 was determined by Western blot analysis. B The amount of Erk-2
(
)
present was determined as a loading control from cells that were untreated Control ; treated for 20 min with S1P, PD98059
( )
(
)
(
)
PD , or forskolin Fors ; or transfected with RasN17. C The cells were treated without or with 50 lM forskolin for 30 min and
stimulated with 1 lM S1P for the indicated times. Raf kinase activity was determined by the immune complex kinase assay with
(
)
unphosphorylated MEK-1 as a substrate upper panel . The amount of immunoprecipitated Raf was determined by Western blot
) ( )
(
(
)
analysis lower panel . D Expression of RasN17 in the cells transfected with an empty vector RasN17 or with an expression
¡
(
)
+
vector for RasN17 RasN17 was determined by Western blot analysis with antibody recognizing both Ras and RasN17 proteins
upper panel . Erk-2 protein was determined by Western blot analysis as a loading control lower panel .
(
)
(
)
( )
Figure 3. EGF receptor tyrosine kinase mediates S1P-induced Erk-1 and -2 activation in Rat-2 cells. A The cells were treated
with vehicle alone or 250 nM of AG1478 for 1 h and were stimulated with either 1 lM S1P or 10 ng/ml EGF for the indicated
( )
times. B The cells were treated with the indicated concentrations of AG1478 for 1 h and then were stimulated with 1 lM S1P or
( )
10 ng/ml EGF for 10 min. Activation of Erk-1/-2 was determined by Western blot analysis. C Erk-2 was determined as a loading
(
)
(
)
control from cells that were untreated Control or treated for 20 min with S1P, EGF, or AG1478 AG .

123
SPHINGOSINE 1-PHOSPHATE ACTIVATION OF ERK-1/-2
( )
blocked the PTX-insensitive Erk-1/-2 activation 6 . Why PKC
has contrasting effects on Erk-1/-2 activation in different cell
types is still uncharacterized; presumably the downstream sig-
naling targets of PKC make the difference. Because PKC has a
(
)
inhibitory role in transactivating EGF receptors 24, 27 , PKC
probably acts as a negative regulator when the EGF receptor
mediates Erk-1/-2 signaling.
In summary, we demonstrated in this study that exogenous
S1P activated Erk-1/-2 in Rat-2 cells by stimulating PTX-sensi-
tive G proteins that caused transactivation of EGF receptor and
subsequent activation of the Ras/Raf/MEK/Erk signaling cas-
cade. S1P-induced transactivation of the EGF receptor appeared
to include PKC as a negative regulator.
Figure 4. S1P-induced transactivation of the EGF receptor is
–
( )
celltype speciŽ c.HeLa,NIH3T3, andSK-N-BE2 C cellswere
treated with vehicle alone or 250 nM of AG1478 for 1 h and
then were stimulated with either 1 l M S1P or 10 ng/ml EGF
for the indicated times. Activation of Erk-1/-2 was determined
by Western blot analysis.
ACKNOWLEDGEMENTS
This work was supported by grants from the Korea Min-
istry of Science and Technology Molecular Medicine Research
(
)
(
Group Program: 99-MM-02-04-A-01 and KOSEF The Inter-
)
disciplinary Research Project:1999-2-207-004-5 .
unmasks the EGF receptor activation. Indeed, our results are in
good agreement with previous reports that inhibition of PKC un-
–
masked EGF receptor activation as a result of G protein coupled
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