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Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA; and the
* Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA
1Correspondence: Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 353 Basic Science Building, 3900 Reservoir Road, NW, Washington, DC 20007, USA. E-mail: spiegel{at}bc.georgetown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
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, phosphatidylinositol
3-kinase, Ras GTPase-activating protein, or protein tyrosine
phosphatase SHP-2, not only failed to increase DNA synthesis in
response to PDGF but also did not activate sphingosine kinase.
Moreover, mutation of tyrosine-1021 of the PDGF receptor to
phenylalanine, which impairs its association with PLC, abrogated
PDGF-induced activation of sphingosine kinase. In contrast, PDGF was
still able to stimulate sphingosine kinase in cells expressing the PDGF
receptor mutated at tyrosines 740/751 and 1009, responsible for binding
of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement,
PDGF did not stimulate sphingosine kinase activity in F5 receptor
`add-back' mutants in which association with the Ras
GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was
individually restored. However, a mutant PDGF receptor that was able to
bind PLC (tyrosine-1021), but not other signaling proteins, restored
sphingosine kinase sensitivity to PDGF. These data indicate that the
tyrosine residue responsible for binding of PLC is required for
PDGF-induced activation of sphingosine kinase. Moreover, calcium
mobilization downstream of PLC, but not protein kinase C activation,
appears to be required for stimulation of sphingosine kinase by
PDGF.—Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel,
S. Platelet-derived growth factor-induced activation of sphingosine
kinase requires phosphorylation of the PDGF receptor tyrosine residue
responsible for binding of PLC.
Key Words: platelet-derived growth factor • SPP • DNA synthesis • DMS
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
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, 2)
. The three
isoforms of PDGF (PDGF-AA, -BB, or -AB) bind with different affinities
to two related tyrosine-kinase receptors, denoted PDGF
and ß
receptors. Dimerization of the receptor leads to activation of the
intrinsic tyrosine kinase activity (1
, 2)
, resulting in
autophosphorylation of the receptor on several tyrosine residues.
Phosphorylation of tyrosine-857 located in tyrosine kinase domain 2
regulates the catalytic activity of the receptor. Other phosphorylated
tyrosine residues located in the juxtamembrane domain, the kinase
insert, and the carboxyl-terminal tail of the PDGF receptor constitute
specific binding sites for multiple intracellular targets that
initiate, directly or indirectly, distinct downstream signaling
cascades (2)
. Some of these targets have been identified,
such as phospholipase C (PLC), phosphatidylinositol-3-kinase
(PI-3 kinase), Ras GTPase-activating protein, Src family kinases,
and Grb2, Nck, Shc adaptor proteins (2)
. Each recognizes
specific tyrosine-phosphate residues in the context of three adjacent
carboxyl-terminal amino acid residues and, in some cases, become
tyrosine-phosphorylated and initiate a signal thereafter.
Normal and mutated PDGF-receptors have been expressed in cells that
lack the PDGF receptor, such as the canine kidney epithelial cell line
TRMP (3)
. By constructing site-directed mutant receptors
with phenylalanine residues instead of specific tyrosine residues, it
was possible to delineate the involvement of the particular signal
transduction molecules that bind to those tyrosine residues and their
correlation with cell proliferation. Thus, it was found that Ras
activation is insufficient to trigger PDGF-dependent DNA synthesis and
that PLC and PI-3 kinase are independent downstream mediators of
PDGF-induced mitogenesis (4)
. However, the precise
involvement of each of these pathways remains unresolved, due in part
to the redundancy in mitogenic signaling induced by PDGF (2
, 4)
.
Data from our laboratory have suggested that sphingosine-1-phosphate
(SPP), a breakdown product of membrane sphingolipids, is another second
messenger involved in the mitogenic response to PDGF (5)
.
Activation of sphingosine kinase, the enzyme that catalyzes the
phosphorylation of sphingosine to SPP, may initiate additional
mitogenic signaling pathways, as SPP has been shown to induce calcium
release from internal stores in an inositol trisphosphate
(InsP3) -independent manner (6)
,
stimulate phospholipase D (7)
, and activate the
extracellular signal-regulated kinase (ERK) cascade (8
, 9)
leading to gene expression (10)
. In addition to PDGF,
other mitogens, including protein kinase C activators (11
, 12)
, the B subunit of cholera toxin (13)
, and other
physiological stimuli including nerve growth factor F (14
, 15)
, vitamin D3 (16)
,
cross-linking of Fc
RI (17)
and FcRI
(18)
, and binding of carbachol to m2 and m3 muscarinic
acetylcholine receptors (19)
, increase cellular levels of
SPP by activation of sphingosine kinase. Most important, both
D,L-threo-dihydrosphingosine (D,L-threo-DHS) and
N,N-dimethylsphingosine (DMS), competitive
inhibitors of sphingosine kinase (20)
, prevented formation
of SPP and not only inhibited cellular proliferation induced by PDGF,
but also blocked calcium mobilization (17
, 19)
and the
cytoprotective effects of survival factors (9
, 20)
,
further supporting a role for endogenous SPP. D,L-threo-DHS
also inhibited the activation of two cyclin-dependent kinases
(p34Cdc2 kinase and Cdk2 kinase) induced by PDGF,
but not by epidermal growth factor (EGF) (21)
. SPP
reversed the inhibitory effects of D,L-threo-DHS,
demonstrating that its effects were mediated via inhibition of
sphingosine kinase. Examination of the early signaling events of PDGF
action revealed that D,L-threo-DHS did not affect
PDGF-induced autophosphorylation of the growth factor receptor or
phosphorylation of the SH2/SH3 adaptor protein Shc and its association
with Grb2, but did inhibit PDGF-stimulated, and not EGF-stimulated, Crk
phosphorylation and ERK activation (21)
. These results
suggest that regulation of sphingosine kinase activity and elevation of
endogenous SPP define divergence in signal transduction pathways of
PDGF and EGF receptors leading to ERK activation.
Despite the importance of sphingosine kinase in cell growth and
survival, little is known about its regulation. To study the mechanisms
of activation of sphingosine kinase by PDGF, we examined PDGF-dependent
sphingosine kinase activation in TRMP cells expressing wild-type (WT)
or mutant PDGFß receptors (ßPDGFR) lacking specific tyrosine
residues and also a series of `add-back' mutants that can selectively
couple to PLC, PI3K, rasGAP, or the protein tyrosine phosphatase
SHP-2 (previously called Syp, SH-PTP2, PTP1D). We found that the
tyrosine residue responsible for binding of PLC (tyrosine-1021) is
required for PDGF-induced activation of sphingosine kinase, which
occurs in a protein kinase C-independent, calcium
mobilization-dependent manner.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
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-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione) were
from Biomol Research Laboratory Inc. (Plymouth Meeting, Pa.).
[-32P]ATP (3000 Ci/mmol) was purchased from
Amersham (Arlington Heights, Ill.).
12-O-Tetradecanoylphorbol-13-acetate (TPA) and essentially fatty
acid-free bovine serum albumin (BSA) were obtained from Sigma (St.
Louis, Mo.). Thapsigargin, BAPTA-AM, and bisindolylmaleimide were from
Calbiochem (San Diego, Calif.). Anti-phosphotyrosine antibody was
obtained from Transduction Laboratories (Lexington, Ky.).
Cells
TRMP canine kidney epithelial cells were infected with a virus
harboring a vector (pLXSN) carrying wild-type ßPDGFR, receptors
mutated at specific tyrosine residues to phenylalanine, or a
kinase-defective PDGF receptor mutant and selected in G418 (1 mg/ml)
medium as described previously (3
, 4)
. This study is
restricted to ßPDGFR, which dimerizes in response to binding PDGF-BB.
Parental TRMP cells do not express detectable levels of the PDGFß
subunit receptor, and cells carrying wild-type ßPDGFR express
~6 x 106 receptors/cell as determined by
quantitative Western immunoblotting (3)
. TRMP cells
express very low levels of PDGFR. We also used a previously
characterized series of PDGFR phosphorylation site mutants that
included F5 (containing tyrosine to phenylalanine substitutions at
positions 740, 751, 771,1009, and 1021) and four `add-back'
constructs in which the binding site for one of the receptor-associated
proteins was added back to the F5 receptor. These mutants are
designated Y740/51, Y771, Y1009, and Y1021, and selectively associate
with PI3K, RasGAP, SHP-2, and PLC, respectively. Cells were cultured
in DMEM supplemented with 4 mM L-glutamine, 10% fetal bovine serum,
100 U/ml penicillin, 100 µg/ml streptomycin and incubated at 37°C
in a humidified, 5% CO2-controlled incubator
(4)
.
DNA synthesis
TRMP cells were seeded in 24-well plates at 3 x
104 cells per well, cultured for 24 h, and
then growth-arrested for 48 h by replacement of the culture medium
with medium containing 2% horse serum. Cells were stimulated with PDGF
or SPP for 18 h and pulsed with 1 µCi of
[3H]thymidine for 2 h (3)
.
Incorporation of radioactivity into trichloroacetic acid-insoluble
material was measured as described (5)
. Values are the
means of triplicate determinations and standard deviations were
routinely less than 10% of the mean.
Sphingosine kinase activity
Sphingosine kinase activity in the cytosol was measured as
described previously (5)
. Briefly, TRMP cells (6 x
105 cells per 100 mm dish) were washed with
phosphate-buffered saline (PBS), serum-starved for 2 days, preincubated
with 50 µM sodium orthovanadate for 30 min, and stimulated with PDGF
for various periods. Cells were washed with PBS, lysed by
freeze-thawing, and cytosolic fractions were prepared by
ultracentrifugation at 105,000 x g for 90 min.
Sphingosine kinase activity was measured by incubating cytosolic
extracts (30 µg) with [32P]ATP (0.5 µCi,
1 mM) containing MgCl2 (10 mM) and 50 µM
sphingosine (BSA complex) in buffer A (20 mM Tris (pH 7.4), 20%
glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate,
40 mM ß-glycero-phosphate, 15 mM NaF, 10 µg/ml leupeptin, aprotinin
and soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride
(PMSF), and 0.5 mM 4-deoxypyridoxine) and incubated for 15 min at
37°C. Labeled lipids were extracted and resolved by thin-layer
chromatography (TLC); [32P]SPP was excised from
the TLC plate and counted by liquid scintillation spectrometry or,
alternatively, quantified with a Molecular Dynamics Storm
PhosphorImager (Sunnyvale, Calif.). Sphingosine kinase specific
activity is expressed as pmol of SPP formed per min/mg protein. Values
are the means of triplicate determinations and standard deviations were
routinely less than 10% of the mean.
Western blot analysis
Tyrosine receptor phosphorylation was analyzed by immunoblotting
with an anti-phosphotyrosine antibody (4)
. Briefly,
serum-starved confluent and quiescent cells were treated with PDGF for
5 min. Cells were then washed and lysed with a buffer consisting of 1%
Triton X-100 in 50 mM HEPES (pH 7.9), 100 mM NaCl, 10 mM EDTA, and a
mixture of phosphatase and protease inhibitors (10 mM NaF, 2 mM sodium
orthovanadate, 4 mM sodium pyrophosphate, 1 mM PMSF, 5 µg/ml
aprotinin, and leupeptin). Lysed cells were harvested by scraping and
centrifuging at 14,500 x g for 10 min at 4°C. The
lysates were boiled in 1x Laemmli sample buffer and equal amounts of
protein separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide
gel electrophoresis) on 7.5% gels. After electrophoretic transfer to
nitrocellulose membranes (MSI, Inc.) using a Bio-Rad transblot
apparatus, the phosphotyrosine containing proteins were detected by
Western blotting using an anti-phosphotyrosine antibody conjugated to
horseradish peroxidase (RC20H) and visualized using enhanced
chemiluminescence reagents.
PKC assay
Cells were washed twice with PBS, scraped from dishes in buffer
A, and lysed by freeze-thawing three times. After centrifugation at
14,000 x g for 20 min, supernatant was saved as the
cytosolic fraction and the pellet was resuspended by passing through a
27-gauge needle 10 times in buffer A containing 0.1% Triton X-100;
protein kinase C (PKC) activity in both fractions was measured using a
PKC assay kit (Upstate Biotechnology, Inc., Lake Placid, N.Y.) as
described previously (20)
.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
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, 4)
. The
advantage of TRMP cells is that though they lack ßPDGFR, exogenously
added SPP can stimulate their growth, suggesting that they contain the
machinery necessary to respond to SPP (Table 1
). As expected, PDGF did not stimulate DNA synthesis or sphingosine
kinase activity in cells carrying an empty viral vector. However, when
these cells were infected with virus encoding the wild-type ßPDGFR
(WT), PDGF stimulated proliferation and concomitantly activated
sphingosine kinase (Table 1)
. The increase in sphingosine kinase
activity induced by PDGF was transient, reaching a maximum within 5–10
min; it remained elevated for 30 min and declined thereafter
(Fig. 1A
). This time course of activation was similar to that
previously observed in Swiss 3T3 fibroblasts (5)
,
which express endogenous ßPDGFR. In more than 20 experiments, basal
activity was in the range of 15–30
pmol/(pmol·min-1·mg-1).
Although PDGF always stimulated sphingosine kinase activity within 10
min in WT cells, the magnitude of the response varies between 1.4- and
1.7-fold stimulation among the different experiments. This is probably
due to slight differences in culture conditions, cell density, and
passage number.
|
|
Similar to our previous studies (5
, 20
, 21)
, inhibition of
sphingosine kinase with DMS partially inhibited the proliferative
effect of PDGF in PDGFR-transfected TRMP cells, suggesting that PDGF
stimulates DNA synthesis by a mechanism that depends in part on
sphingosine kinase activation (Fig. 1B
).
TRMP cells that express ßPDGFR with arginine at position 635
(R635) and are tyrosine kinase defective (KD-PDGFR) (22)
failed to stimulate sphingosine kinase or DNA synthesis in response to
PDGF. The kinase inactive receptor is able to dimerize upon binding of
PDGF-BB but is not able to autophosphorylate (Fig. 2
B). These results suggest that the tyrosine kinase activity
of the receptor is essential for sphingosine kinase activation by PDGF.
In agreement, genistein, a general inhibitor of tyrosine
phosphorylation that greatly inhibits the tyrosine kinase activity of
the receptor, completely abolished the activation of sphingosine kinase
by PDGF-BB in wild-type ßPDGFR-expressing cells (data not shown).
Sphingosine kinase activation by a nongrowth factor receptor, FcRI,
is also tyrosine kinase dependent (18)
.
|
TRMP cells expressing mutated PDGFR (F5 mutants) with phenylalanine
substitutions at five major tyrosine phosphorylation sites
(740/751/771/1009/1021), which are therefore unable to associate with
PLC, PI3K, Ras-GAP, or SHP-2, also failed to increase DNA synthesis
(4)
and activate sphingosine kinase in response to PDGF
(Table 1)
. It should be noted that the F5 mutant is efficiently
phosphorylated in response to PDGF (Fig. 2B
). This has been
observed in TRMP and other cell types (4
, 22
23
24)
, and may
reflect the fact that this receptor retains a number of phosphorylation
sites, including a major one at Y857. Moreover, the nonreceptor
tyrosine kinase Src is able to associate with the juxtamembrane domain,
tyrosine residues 579/581 (25)
present in all of the PDGFR
described here. Our results suggest that activation of sphingosine
kinase by PDGF depends on the phosphorylation of one or more of the
specific tyrosines mutated in the F5 PDGFR mutants and might be
downstream of PLC, PI3K, Ras-GAP, and/or SHP-2 signaling.
To determine which tyrosine residue is responsible for the activation
of sphingosine kinase, we used PDGFR selectively mutated in only one or
two tyrosines. Cells expressing PDGFR mutated in positions 740/751
(F740/751) and 1009 (F1009), which fail to associate with PI3K and
SHP-2, respectively, still retained the ability to stimulate
sphingosine kinase when activated by PDGF-BB (Fig. 2A
). In
contrast, F1021, which is impaired in its association with PLC, was
unable to activate sphingosine kinase (Fig. 2A
). These
mutants all had intact PDGFR tyrosine kinase activity because they were
phosphorylated to a similar extent as the wild-type receptor (Fig. 2B
). Thus, it seems that the tyrosine kinase activity of the
PDGFR per se is not sufficient for activation of sphingosine
kinase and that autophosphorylation of the receptor on the specific
tyrosine residue responsible for its association with PLC is
critical.
The PLC binding site of PDGFR is required for stimulation of
sphingosine kinase by PDGF
To further assess the role of tyrosine-1021 in activation of
sphingosine kinase by PDGF-BB and to study whether cross talk with
other signaling pathways also contributes to its activation, we used
`add back' mutants (4)
, another series of mutants that
are obtained by restoring individual tyrosine residues of the F5 mutant
(4)
. We found that replacement of only phenylalanine in
position 1021 by tyrosine is sufficient to restore most of the
activation of sphingosine kinase by PDGF, indicating that the absence
of the remainder of the other signals is not crucial for this response
(Fig. 3
). In agreement with previous reports (4
, 22
, 23)
, mutation
of tyrosine-1021 greatly impaired the ability of PDGF to induce
proliferation (data not shown). Although phosphorylation of
tyrosine-1021 is specifically required for association with PLC, it
is also possible that other proteins bind to the same site on the
receptor. Thus, if sphingosine kinase associates directly or indirectly
with phosphorylated tyrosine-1021 upon activation, it should
coimmunoprecipitate with PDGFR. However, immunoprecipitation of lysates
from cells expressing wild-type PDGFR with phospho-tyrosine antibody
did not coprecipitate sphingosine kinase activity after treatment of
the cells with PDGF, suggesting that sphingosine kinase may not be a
substrate of the PDGFR tyrosine kinase and that its activation does not
involve physical association with PDGFR.
|
Calcium mobilization but not activation of PKC is required for
PDGF-induced stimulation of sphingosine kinase
Another possibility is that activation of sphingosine kinase
occurs as a consequence of a downstream signal of PLC. PLC
activation results in the formation of the second messenger
InsP3, which mobilizes calcium from internal
sources, and diacylglycerol (DAG), which activates PKC. U73122 is
generally used as a selective inhibitor of PLC and the related rise in
cytosolic free Ca2+. Pre-exposure of cells to
U73122 abolished activation of sphingosine kinase induced by PDGF
(Fig. 4
A), suggesting that PLC activity may be required.
Surprisingly, U73343, an inactive analog, also inhibited
PDGF-stimulated sphingosine kinase activity (data not shown).
Similarly, it has recently been shown that U73122 and U73343 inhibit
receptor-mediated PLD activation downstream of PLC in CHO cells
(26)
. Moreover, it has been suggested that the effect of
U73122 may be mediated by effects on calcium, since U73122 is more
potent in antagonizing Ca2+ channels:
intracellular channels, which are activated by formation of
InsP3, and those present on the plasma membrane
(27)
.
|
A useful approach to study the involvement of intracellular calcium
sources is to deplete the pools by a mechanism independent of receptor
activation and the generation of InsP3, such as
inhibiting calcium-sequestering ATPases. Thapsigargin is an
irreversible inhibitor of the endoplasmic reticulum
Ca2+-ATPase, but does not inhibit the
plasmalemmal enzyme and is frequently used to deplete select
intracellular calcium pools (28)
. Thapsigargin itself
stimulated sphingosine kinase activity, probably due to its ability to
induce a slow increase in
[Ca2+]i (6)
.
However, there was no further increase in sphingosine kinase activity
in the presence of PDGF (Fig. 4B
). The binding of
intracellular free Ca2+ by BAPTA-AM inhibited
PDGF-stimulated sphingosine kinase activity (Fig. 4B
),
suggesting that a rapid rise in cytosolic free calcium plays an
important role in stimulation of sphingosine kinase activity. In
agreement, calcium ionophores stimulate sphingosine kinase activity
(data not shown).
Calcium mobilization, together with DAG, contributes to the activation
of cPKC (29)
. PKC has been shown to activate sphingosine
kinase with concomitant increases in SPP levels in a number of cell
systems (9
, 11
, 12
, 16
, 20)
. Moreover, inhibition of PKC
markedly reduced sphingosine kinase activity and the apoptosis-sparing
effect of vitamin D3 (16)
.
Therefore, activation of sphingosine kinase by PDGF might also involve
PKC. Prolonged exposure of cells to TPA, a functional analog of DAG,
down-regulates the PKC isoforms that are stimulated by DAG while having
a much smaller effect on DAG-insensitive PKC family members
(29)
. Chronic treatment with TPA had only a small effect
on the ability of PDGF to stimulate sphingosine kinase (Fig. 5
A), but completely eliminated the ability of TPA to activate
sphingosine kinase (data not shown). In contrast, it has previously
been shown that TPA pretreatment in these cells led to a complete loss
of PDGF-activated phospholipase D activity (30)
. We also
used a panel of commercially available inhibitors of PKC, including the
bisindolylmaleimide GF 109203X,
1-(5-isoquinolinysulfonyl)-2-methylpiperazine (H7), chelerythrine
chloride, and calphostin C. When cells were treated with these
inhibitors before PDGF stimulation, sphingosine kinase was still
activated (Fig. 5B
), whereas they blocked sphingosine kinase
activation induced by TPA (data not shown). Although H7 inhibits other
kinases as well as PKC, the other inhibitors appear to be more specific
for PKC. Moreover, bisindolylmaleimide, which shows high selectivity
for calcium- and DAG-dependent PKCs (cPKC), did not impair the
activation of sphingosine kinase by PDGF, but completely blocked the
activation of sphingosine kinase by TPA (Fig. 5C
) and
TPA-induced activation of PKC (Fig. 5D
). Thus, activation of
cPKC may not be required for PDGF to stimulate sphingosine kinase.
|
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CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
|---|
(tyrosine-1021) to ßPDGFR is required for PDGF-induced
activation of sphingosine kinase, the enzyme that regulates the levels
of the intracellular second messenger SPP. Downstream of PLC-,
calcium mobilization (but not PKC activation) appears to be required
for stimulation of sphingosine kinase by PDGF. Tyrosine-1021 has also
been shown to be required for PDGF-induced activation of PLD leading to
formation of another lipid second messenger, phosphatidic acid; but in
contrast to sphingosine kinase activation, phosphatidic acid formation
was dependent on PKC activation (30)
. Thus, it is not
surprising that this residue is critically important for cellular
proliferation and transformation induced by PDGF (22
, 23)
,
since it is important for formation of a myriad of second messengers.
Although PLC activation is sufficient by itself to induce cellular
proliferation, PKC might not be involved since TPA does not stimulate
proliferation of TRMP cells, yet markedly stimulates PKC
(4)
. The results presented here suggest that SPP might be
the missing link between activation of PLC and cellular
proliferation induced by PDGF. Since SPP itself can release calcium
from intracellular sources, leading to an influx of extracellular
calcium, sphingosine kinase activation may represent a mechanism of
amplification of calcium-related signals to ensure long-term cell
proliferation. SPP induces calcium release from both
InsP3-sensitive and -insensitive pools
(6)
, thereby further enhancing calcium mobilization in
response to PDGF. In support of this conclusion, a recent study
demonstrated that PDGF-induced calcium entry does not require
InsP3-mediated release of intracellular calcium
stores and that PDGF causes substantially more calcium entry than
InsP3 alone (31)
. Both calcium entry
and intracellular calcium release were absent in Chinese hamster ovary
cells expressing a PDGFR mutant that fails to bind PLC
(31)
. These results suggest the existence of a second
messenger downstream of PLC other than InsP3
that probably plays a role in PDGF-mediated calcium entry. It is
tempting to speculate that SPP might be this additional second
messenger. Furthermore, a recent study demonstrated that sphingosine
directly blocks calcium release-activated calcium current (ICRAC)
(32)
, and it has been suggested that upon mobilization of
internal calcium stores, conversion of sphingosine to SPP by activation
of sphingosine kinase lowers sphingosine levels and leads to the
disinhibition of ICRAC. The recent cloning of sphingosine kinase
(33)
should help in understanding cross talk between
different signaling pathways regulated by PDGF that are important for
calcium homeostasis and cellular proliferation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication December 16, 1998. Revised for publication March 29, 1999.
|
REFERENCES |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSREFERENCES |
|---|
1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell 73,321-324[Medline]
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RI coupling to phospholipase D initiates sphingosine kinase-mediated calcium mobilization and vesicular trafficking. J. Biol. Chem. 273,9393-9402 or phosphatidylinositol 3 kinase. J. Biol. Chem. 272,9011-9018 is necessary for stimulation of phospholipase D by platelet-derived growth factor. J. Biol. Chem. 269,27823-27826This article has been cited by other articles:
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  V. Anelli, C. R. Gault, A. B. Cheng, and L. M. Obeid Sphingosine Kinase 1 Is Up-regulated during Hypoxia in U87MG Glioma Cells: ROLE OF HYPOXIA-INDUCIBLE FACTORS 1 AND 2 J. Biol. Chem., February 8, 2008; 283(6): 3365 - 3375. [Abstract] [Full Text] [PDF]
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J. Radeff-Huang, T. M. Seasholtz, J. W. Chang, J. M. Smith, C. T. Walsh, and J. H. Brown Tumor Necrosis Factor-{alpha}-stimulated Cell Proliferation Is Mediated through Sphingosine Kinase-dependent Akt Activation and Cyclin D Expression J. Biol. Chem., January 12, 2007; 282(2): 863 - 870. [Abstract] [Full Text] [PDF]
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 B. Soliven, L. Ma, H. Bae, B. Attali, A. Sobko, and T. Iwase PDGF upregulates delayed rectifier via Src family kinases and sphingosine kinase in oligodendroglial progenitors Am J Physiol Cell Physiol, January 1, 2003; 284(1): C85 - C93. [Abstract] [Full Text] [PDF]
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X. Shu, W. Wu, R. D. Mosteller, and D. Broek Sphingosine Kinase Mediates Vascular Endothelial Growth Factor-Induced Activation of Ras and Mitogen-Activated Protein Kinases Mol. Cell. Biol., November 15, 2002; 22(22): 7758 - 7768. [Abstract] [Full Text] [PDF]
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E. Lacana, M. Maceyka, S. Milstien, and S. Spiegel Cloning and Characterization of a Protein Kinase A Anchoring Protein (AKAP)-related Protein That Interacts with and Regulates Sphingosine Kinase 1 Activity J. Biol. Chem., August 30, 2002; 277(36): 32947 - 32953. [Abstract] [Full Text] [PDF]
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L. R. Vann, S. G. Payne, L. C. Edsall, S. Twitty, S. Spiegel, and S. Milstien Involvement of Sphingosine Kinase in TNF-alpha -stimulated Tetrahydrobiopterin Biosynthesis in C6 Glioma Cells J. Biol. Chem., April 5, 2002; 277(15): 12649 - 12656. [Abstract] [Full Text] [PDF]
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K. W. Young, M. D. Bootman, D. R. Channing, P. Lipp, P. R. Maycox, J. Meakin, R. A. J. Challiss, and S. R. Nahorski Lysophosphatidic Acid-induced Ca2+ Mobilization Requires Intracellular Sphingosine 1-Phosphate Production. POTENTIAL INVOLVEMENT OF ENDOGENOUS EDG-4 RECEPTORS J. Biol. Chem., December 1, 2000; 275(49): 38532 - 38539. [Abstract] [Full Text] [PDF]
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I. Kim, S.-O. Moon, S. Hoon Kim, H. Jin Kim, Y. Soon Koh, and G. Young Koh Vascular Endothelial Growth Factor Expression of Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and E-selectin through Nuclear Factor-kappa B Activation in Endothelial Cells J. Biol. Chem., March 2, 2001; 276(10): 7614 - 7620. [Abstract] [Full Text] [PDF]
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C. J. Birchwood, J. D. Saba, R. C. Dickson, and K. W. Cunningham Calcium Influx and Signaling in Yeast Stimulated by Intracellular Sphingosine 1-Phosphate Accumulation J. Biol. Chem., April 6, 2001; 276(15): 11712 - 11718. [Abstract] [Full Text] [PDF]
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