From the Department of Biology, Johns Hopkins
University, Baltimore, Maryland 21218, the § Children's
Hospital Oakland Research Institute, Oakland, California 94609, and the
¶ Department of Biochemistry, University of Kentucky College of
Medicine, Lexington, Kentucky 40536
Received for publication, November 9, 2000, and in revised form, January 17, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mammalian cells, intracellular sphingosine
1-phosphate (S1P) can stimulate calcium release from intracellular
organelles, resulting in the activation of downstream signaling
pathways. The budding yeast Saccharomyces cerevisiae
expresses enzymes that can synthesize and degrade S1P and related
molecules, but their possible role in calcium signaling has not yet
been tested. Here we examine the effects of S1P accumulation on
calcium signaling using a variety of yeast mutants. Treatment of yeast
cells with exogenous sphingosine stimulated Ca2+
accumulation through two distinct pathways. The first pathway required
the Cch1p and Mid1p subunits of a Ca2+ influx channel,
depended upon the function of sphingosine kinases (Lcb4p and Lcb5p),
and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P
phosphatase (Lcb3p). The biologically inactive stereoisomer of
sphingosine did not activate this Ca2+ influx pathway,
suggesting that the active S1P isomer specifically stimulates a
calcium-signaling mechanism in yeast. The second Ca2+
influx pathway stimulated by the addition of sphingosine was not
stereospecific, was not dependent on the sphingosine kinases, occurred
only at higher doses of added sphingosine, and therefore was likely to
be nonspecific. Mutants lacking both S1P lyase and phosphatase
(dpl1 lcb3 double mutants) exhibited constitutively high
Ca2+ accumulation and signaling in the absence of added
sphingosine, and these effects were dependent on the sphingosine
kinases. These results show that endogenous S1P-related molecules can
also trigger Ca2+ accumulation and signaling. Several
stimuli previously shown to evoke calcium signaling in wild-type cells
were examined in lcb4 lcb5 double mutants. All of the
stimuli produced calcium signals independent of sphingosine kinase
activity, suggesting that phosphorylated sphingoid bases might serve as
messengers of calcium signaling in yeast during an unknown cellular response.
Sphingolipid metabolites, such as ceramide, sphingosine, and
S1P,1 function as important
second messengers in mammalian cells mediating processes such as cell
proliferation and motility, differentiation, senescence, stress
responses, and apoptosis (1). S1P accumulates in response to
various physiological stimuli in mammals. In RBL-2H3 cells, for
example, the clustering of the IgE receptor Fc Sphingolipids are abundant components of the plasma membrane in yeast,
comprising 30% of total membrane phospholipids (6). They differ
slightly from mammalian sphingolipids, using a derivative of
sphingosine known as phytosphingosine. The enzymes in yeast responsible
for phosphorylation of endogenous phytosphingosine and exogenous long
chain bases such as sphingosine have recently been identified. Two
related sphingosine kinases were identified in yeast as the products of
the LCB4 and LCB5 genes (7, 8). Mutants lacking
both sphingosine kinases accumulate no detectable S1P-related molecules
but, nevertheless, are viable and exhibit no obvious phenotypes (7, 9).
Yeast also expresses two related S1P phosphatases encoded by the
LCB3/YSR2 and YSR3 genes, the former being the
major enzyme (10-12). Additionally, yeast cells express S1P lyase
encoded by DPL1 (formerly BST1), which cleaves S1P to yield ethanolamine-1-phosphate and hexadecanal (13). Mutants lacking S1P lyase (dpl1/bst1 mutants) accumulate
phyto-S1P and dihydro-S1P at slightly elevated levels (9, 14) and reach maximal S1P accumulation levels within 60 min of the addition of
sphingosine to the culture medium (13). These effects are further
exacerbated in dpl1 lcb3 double mutants lacking both S1P lyase and the major S1P phosphatase with 500 times greater levels of
phyto-S1P and dihydro-S1P relative to wild type (7, 14). Although the
functions of S1Ps in yeast are largely unknown, recent evidence
suggests a role for these lipids in resistance to heat stress in the
regulation of cell proliferation and in the shift from fermentative to
respiratory growth (8, 14-16). The possibility that S1Ps regulate
calcium signaling in yeast cells has not been examined.
In yeast, calcium signals are generated in response to a variety of
external stimuli including mating pheromones, salt stress, glucose-1-phosphate accumulation, and depletion of Ca2+
from secretory organelles (17-21). Yeast expresses a Ca2+
influx channel related to voltage-gated Ca2+ channels of
animals (22-24) in addition to various intracellular Ca2+
pumps and exchangers related to animal enzymes (25-29). However, homologs of the sarcoendoplasmic reticulum calcium ATPase-type Ca2+ pump and the inositol 1,4,5-trisphosphate
(IP3) or ryanodine receptors, which supply and release
Ca2+ from the endoplasmic reticulum in animal cells, are
not evident in the yeast genome. Nevertheless, the yeast endoplasmic
reticulum accumulates sufficient Ca2+ to facilitate protein
folding and secretion, in part through Pmr1p, a member of the secretory
pathway calcium ATPase family (21, 29). Sequences of the
mammalian S1P-receptor involved in Ca2+ release from
microsomes have not been reported.
Here we show that conversion of exogenously added sphingosine to
sphingosine 1-phosphate stimulates Ca2+ influx,
accumulation, and signaling in yeast. Similar to the CCE-like mechanism
of yeast (15), the calcium channel subunit Cch1p was required for the
majority of S1P-stimulated Ca2+ accumulation. Therefore,
yeast may retain S1P-regulated calcium-signaling mechanisms analogous
to those of mammalian cells.
Yeast Strains and Growth Media--
All yeast strains (Table
I) were maintained on either YPD medium
or synthetic complete medium (SC) lacking leucine or uracil. Strains of
the JK9-3d background CBY31, CBY32, CBY33, and CBY34 were
constructed from MSS200, MSS204, MSS205, and MSS207 (14), respectively,
by curing the TPS2-lacZ::URA3 plasmid
after growth in 5-fluoroorotic acid. CBY79 (dpl1
cch1) was constructed by transformation using the
cch1::TRP1 disruption plasmid pKC289
(24). Similarly, CBY224-229 were constructed by transformation using
the pmr1::LEU2 disruptant plasmid pL119
(30). Finally, pgm2::LEU2 mutant
strains were constructed by transformation of CBY31 and CBY106 using
pDB419 (17). All disruptions were confirmed by polymerase chain
reaction and/or phenotypic analyses. All other strains were constructed by isogenic crosses.
Reagents--
D-Erythro-sphingosine and
L-erythro-sphingosine, phytosphingosine, and
dihydrosphingosine were purchased from Sigma. FK506 was generously
provided by Fujisawa USA, Inc. (Tokyo, Japan). Coelenterazine was
obtained from Molecular Probes, Inc. 45CaCl2
was obtained from Amersham Pharmacia Biotech.
Calcium Accumulation Assays--
Cells were grown overnight at
30 °C in SC medium. Log phase cells were harvested, resuspended in
fresh SC medium to an A600 of 1-2, and
then diluted 2-fold into medium containing
45Ca2+, phytosphingosine, dihydrosphingosine,
sphingosine, chlorpromazine, and/or FK506, as described in the
text. Cells were harvested by filtration, washed, and processed
for determination of total associated Ca2+ as described
previously (25).
Aequorin Luminescence Assays--
Assays were performed as
described previously (31). Cells expressing
pKC1462 apoaequorin were
grown overnight at 30 °C in SC medium lacking uracil. Log phase
cells were concentrated to an A600 of 100 in SC
lacking leucine medium, incubated with 10% v/v of 590 µM
natural coelenterazine stock for 20 min, washed, and diluted 100-fold into fresh medium supplemented with 12.5 µM sphingosine,
10 mM BAPTA, and/or 5 µg/ml cycloheximide. The resulting
luminescence was measured at intervals for 2-3 h. Cells were
subsequently permeabilized with 250 µM digitonin, and the
luminescence was recorded to standardize for aequorin loading between strains.
Sphingosine 1-Phosphate Stimulates Calcium Influx and Signaling in
Yeast--
Exogenous sphingosine added to culture medium can be taken
up by yeast cells and phosphorylated to S1P by sphingosine kinases (13). S1P can be dephosphorylated by the phosphatase Lcb3p or degraded
by the lyase Dpl1p (11-13). To determine whether S1P accumulation can
evoke calcium signaling in yeast, we first monitored the accumulation of 45Ca2+ from the medium into growing yeast
cells treated with a wide range of exogenous sphingosine. The addition
of sphingosine to the culture medium at concentrations >25
µM stimulated 45Ca2+ accumulation
in wild-type yeast strains up to 2× the basal level (Fig.
1A). Mutants lacking the S1P
phosphatase (lcb3 mutants) were indistinguishable from wild
type in this assay. In contrast, dpl1 mutants lacking S1P
lyase accumulated 5-fold higher amounts of
45Ca2+ after treatment with only 10-15
µM sphingosine. These results show that exogenous
sphingosine can stimulate Ca2+ accumulation and that this
response can be inhibited by the S1P lyase Dpl1p.
If phosphorylation of exogenous sphingosine was required to promote
Ca2+ influx, mutants lacking the sphingosine kinases would
exhibit less Ca2+ accumulation after treatment with
sphingosine. Indeed, the 5-fold increase in
45Ca2+ accumulation seen in a dpl1
mutant at 12.5 µM sphingosine was abolished in dpl1
lcb4 lcb5 triple mutants (Fig. 1B, left),
indicating that this dramatic increase in Ca2+ accumulation
was dependent on the S1P produced by Lcb4p and Lcb5p. However, the
lcb4 lcb5 double mutants exhibited wild-type sensitivity to
sphingosine (Fig. 1B, right). Thus, exogenous
sphingosine produced two separable calcium responses in yeast, one that
was relatively small and independent of sphingosine kinases and another
larger response that was dependent on sphingosine kinases and sensitive to S1P lyase and phosphatase. All future experiments will examine the
properties of the latter S1P-specific response, which is prominent in
dpl1 mutants.
Sphingosine kinases typically phosphorylate the natural
D-isomer of sphingosine and are unable to act on the
L-isomer (8). The addition of L-sphingosine to
yeast cultures stimulated 45Ca2+ accumulation
in dpl1 mutants only at very high concentrations similar to
those effective in dpl1 lcb4 lcb5 triple mutants (Fig. 1C). Thus, L-sphingosine failed to stimulate
Ca2+ accumulation through the pathway involving sphingosine
kinases even in the supersensitive dpl1 strain. The results
confirm the existence of two separable responses to added sphingosine,
one that is not stereospecific or dependent on sphingosine kinases and
one that is specific for the biologically active D-isomer of sphingosine, dependent on sphingosine kinases, and sensitive to S1P
lyase and phosphatase.
Yeast and other fungi synthesize phytosphingosine rather than
sphingosine, as well as its precursor dihydrosphingosine. These molecules differ in the level of saturation and hydroxylation at C-4
(35). Exogenous phytosphingosine and dihydrosphingosine stimulated the
nonspecific Ca2+ response much like sphingosine but only
weakly stimulated the specific Ca2+ response in
dpl1 mutants (Fig.
2A). The weaker effects of
exogenous phytosphingosine and dihydrosphingosine might be explained if they are poorer substrates than sphingosine for the kinases (7, 8) or
if their phosphorylated products are better substrates than S1P for
Lcb3p.
The lcb3 single mutants accumulate approximately 10-fold
higher levels of phyto-S1P and dihydro-S1P than wild type during vegetative growth (9, 14), but they do not accumulate more 45Ca2+ than wild type with or without added
sphingosine (Fig. 1A). Therefore, a higher threshold level
of the native S1Ps may be necessary to stimulate Ca2+
influx. To evaluate the possible roles of phyto-S1P and dihydro-S1P more carefully, we measured 45Ca2+ accumulation
into a dpl1 lcb3 double mutant. A dpl1 lcb3
double mutant that is also auxotrophic for certain amino acids is
inviable in standard medium unless sphingosine kinases are also
inactivated (9). However, a prototrophic dpl1 lcb3 double
mutant is viable and accumulates ~500 times higher levels of
phyto-S1P and dihydro-S1P than wild type (14). We observed that the
viable dpl1 lcb3 double mutant accumulated
45Ca2+ from the medium at a constitutively high
rate in the absence of added sphingolipids and also showed even greater
sensitivity to added sphingosine than dpl1 mutants (Fig.
1A). The high rate of 45Ca2+
accumulation in dpl1 lcb3 double mutants was not observed in dpl1 lcb3 lcb4 lcb5 quadruple mutants (Fig. 2B),
which fail to accumulate detectable levels of dihydro-S1P and phyto-S1P
(9). These results confirm that accumulation of S1P-related molecules native to yeast can stimulate 45Ca2+ influx and
accumulation and suggest that Dpl1p inhibits the response more potently
than Lcb3p.
S1P-stimulated Ca2+ Accumulation Involves the
Cch1p-dependent Ca2+ Channel--
Cch1p was
identified previously as the probable pore-forming subunit of a plasma
membrane Ca2+ influx channel that is activated by a variety
of stimuli (22, 23). To determine whether S1P stimulates Cch1p
activity, we monitored Ca2+ accumulation in a
dpl1 mutant and a cch1 dpl1 double mutant after the addition of sphingosine. The dpl1 cch1 double mutant
displayed sensitivity to sphingosine, which was similar to that of the
dpl1 single mutant, but the maximal level of
45Ca2+ accumulation was lower in the cch1
dpl1 double mutant than in the dpl1 single mutant (Fig.
3, A and B). The
residual effect of sphingosine in the cch1 dpl1 double
mutant was not observed in cch1 dpl1 lcb4 lcb5 quadruple
mutants. Therefore, the Cch1p channel was required for the major
component of the S1P-stimulated 45Ca2+
accumulation under these conditions.
S1P Accumulation Elevates [Ca2+]c and
Activates Calcineurin-signaling Pathways--
The kinetics of the
S1P-specific response were monitored using cells expressing the
calcium-sensitive photoprotein aequorin in the cytoplasm. Treatment of
a dpl1 mutant with 12.5 µM sphingosine produced a detectable increase in aequorin luminescence within 60-80
min of treatment that rose sharply and reached a plateau for at least
1 h (Fig. 4, A and
B) (data not shown). Treatment of a dpl1 lcb4
lcb5 triple mutant in a parallel experiment resulted in little
aequorin luminescence over this time frame, indicating that S1P
accumulation elevates cytosolic-free Ca2+
([Ca2+]c) primarily via the specific pathway. The
protein synthesis inhibitor cycloheximide completely abolished the
response of dpl1 mutants to sphingosine (Fig.
4B), suggesting that protein synthesis was necessary for
Ca2+ influx and [Ca2+]c elevation in
response to sphingosine. Aequorin luminescence in dpl1
mutants was also abolished by the addition of the Ca2+ ion
chelator BAPTA to the culture medium, suggesting that the [Ca2+]c elevation observed was the result of
extracellular Ca2+ influx and not intracellular
Ca2+ release (Fig. 4A). In summary, the
specific response to S1P stimulated Ca2+ influx through
Cch1p and elevated [Ca2+]c.
To determine whether [Ca2+]c elevation in
response to S1P could activate downstream signaling pathways, the
expression of a calcineurin-dependent reporter gene
PMC1-lacZ was quantitated in cells treated with sphingosine.
Sphingosine treatment induced the expression of PMC1-lacZ in
a dpl1 mutant but not in dpl1 lcb4 lcb5 triple
mutants (Fig. 5A). The
induction of PMC1-lacZ in dpl1 mutants was
completely blocked by treatment with either FK506, a cell-permeant
inhibitor of calcineurin, or membrane-impermeant BAPTA (Fig.
5B). Therefore, the S1P-specific response activated the
calcineurin-signaling pathway in yeast by a mechanism requiring influx
of extracellular Ca2+. In dpl1 lcb3 double
mutants, PMC-lacZ expression was constitutively high in the
absence of added sphingosine. This expression still required the
function of the sphingosine kinases Lcb4p and Lcb5p and was sensitive
to calcineurin inhibitors (Fig. 5C). Taken together, these
findings demonstrate that accumulation of S1P and related endogenous
molecules can stimulate calcium influx and signaling in yeast.
Do S1Ps Serve as Second Messengers for Calcium
Signaling?--
Several external stimuli lead to the generation of
calcium signals in yeast. Although none of these stimuli has been shown to affect metabolism of S1P-related molecules, it is conceivable that
one or more of these stimuli causes increased accumulation of S1Ps,
which then triggers calcium signaling. To test whether endogenous
S1P-related molecules are required for generating calcium signals in
response to known physiological stimuli, we compared the calcium
responses of wild type or dpl1 mutant cells to those of
lcb4 lcb5 or dpl1 lcb4 lcb5 mutant cells lacking
sphingosine kinases over a wide range of conditions. The first stimulus
tested, The results reported here suggest that accumulation of S1P or
related molecules in yeast can stimulate Ca2+ influx via
Cch1p and other factors, resulting in the elevation of
[Ca2+]c and activation of calcineurin signaling.
Several conditions shown previously to accumulate S1P or the native
derivatives phyto-S1P and dihydro-S1P were found to stimulate
Ca2+ influx and signaling in a manner requiring the
homologous sphingosine kinases Lcb4p and Lcb5p. Dpl1p, the S1P lyase,
potently blocked the Ca2+ influx responses with or without
Lcb3p, the major S1P phosphatase. Lcb3p was less effective in this
regard and only significant in dpl1 mutants, possibly
because of the activity of Ysr3p, a minor S1P phosphatase homologous to
Lcb3p (11). The direct targets or derivatives of S1P that lead to
Ca2+ influx and signaling have not been determined.
However, the 1-h lag time after the addition of sphingosine observed
before the onset of Ca2+ influx and the sensitivity to
cycloheximide suggests that the response might reflect the time
necessary for expressing new proteins involved in Ca2+
influx and sufficient buildup of S1P. In the presence of extracellular Ca2+ chelators such as BAPTA, sphingosine was completely
unable to elevate [Ca2+]c (Fig. 4A).
Therefore, no evidence for S1P-triggered Ca2+ release was
obtained in yeast thus far.
Exogenous D-sphingosine also stimulated Ca2+
accumulation to a smaller degree independent of the sphingosine kinases
Lcb4p and Lcb5p. This kinase-independent response may be
nonphysiological because a similar effect was detected using the
biologically inactive isomer L-sphingosine and because of
the very high doses necessary to achieve the response. We have noticed
similar responses of yeast cells to low levels of other membrane active
compounds such as lyso-phosphatidic acid and
detergents.3
Unlike these nonspecific effects, the involvement of the
Ca2+ channel protein Cch1p and the requirement for S1P
biosynthesis and accumulation underscore the existence of specific
mechanism for S1P-dependent calcium signaling in yeast.
Cch1p activity was stimulated in pmr1 mutants (lacking the
secretory pathway Ca2+ATPase) upon depletion of
Ca2+ from secretory organelles (24). Treatment of mammalian
cells with thapsigargin, a specific inhibitor of sarcoendoplasmic
reticulum calcium ATPase that rapidly depletes Ca2+ from
the endoplasmic reticulum and stimulates CCE mechanisms, was shown to
stimulate sphingosine kinase activity (38). However, it seems
improbable that accumulation of S1Ps serves as a messenger of CCE in
yeast cells, because pmr1 lcb4 lcb5 triple mutants lacking the sphingosine kinases exhibited as much Ca2+ accumulation
as pmr1 mutants (Fig. 6D). Additionally, the
injection of S1P in mammalian RBL cells failed to stimulate the CCE
channel known as ICRAC, and inhibitors of sphingosine
kinase failed to prevent ICRAC activation by thapsigargin
(39). A more reasonable explanation is that accumulation of S1Ps
stimulates CCE in animal and fungal cells by first triggering
Ca2+ release from secretory organelles.
S1P rapidly stimulates Ca2+ release from the endoplasmic
reticulum of mammalian cells followed by stimulation of
Ca2+ influx at the plasma membrane and signaling (3, 5).
The receptor for S1P has not been identified, but evidence suggests the
Ca2+ release channel in the endoplasmic reticulum is
distinct from the well characterized IP3 receptor and
ryanodine receptor (3). All these routes of Ca2+ release
can rapidly deplete the endoplasmic reticulum of Ca2+ and
activate CCE pathways. The yeast genome lacks sequences orthologous to
the IP3 and ryanodine receptors, but nevertheless, yeast
cells may utilize a mechanism resembling CCE to supply Ca2+
to secretory organelles (24). Our data do not rule out the possibility
that S1P activates a new class of Ca2+ release channels in
yeast that is potentially related to the unidentified S1P-receptor in
animal cells. Support for this hypothesis might be obtained through the
identification of new factors required for S1P-stimulated
Ca2+ signaling in yeast.
What is the purpose of S1P-stimulated Ca2+ influx and
signaling in yeast? That yeast would encounter either high sphingosine environments or conditions inactivating both S1P lyase and phosphatase seems improbable. Therefore, we examined a number of previously described stimuli that lead to Ca2+ signaling in wild type
and in lcb4 lcb5 double mutants and found no evidence for
the involvement of S1P or its derivatives in any of the processes. It
is possible that a significant contribution of S1Ps to Ca2+
signaling was masked by the action of functionally redundant pathways,
similar to what has been previously proposed for the sphingosine
kinases in the heat stress response (35). Alternatively, an untested
stimulus may be found that causes S1P accumulation and stimulation of
Ca2+ signaling. It seems probable that the stimulation of
calcium influx and signaling by S1P is the result of a physiological
phenomenon because of the similar responses to S1P in animal cells and
the strong conservation of sphingosine kinases, S1P lyases, and S1P phosphatases among animals, fungi, and plants. Furthermore,
sphingolipid perturbations have previously been shown to affect calcium
homeostasis (32, 40), perhaps indicating cross-regulation of the two systems.
The analysis of yeast mutants lacking S1P synthesis and degradation
factors has produced few insights into their physiological roles.
Mutants lacking Dpl1p and Lcb3p exhibit enhanced tolerance of acute
heat shock, whereas mutants lacking the sphingosine kinases exhibit
slightly enhanced sensitivity (8, 35). The levels of dihydro-S1P and
phyto-S1P also increase transiently (~10 min) after heat shock (14).
However, the degree of heat tolerance or sensitivity conferred by these
mutants varies significantly with the conditions used (35). We observed
a transient elevation of [Ca2+]c after heat
stress, but the kinetics and magnitude were similar in dpl1
and dpl1 lcb4 lcb5 double mutants (Fig. 6C). Thus, the activity of the sphingosine kinases (and hence the ability to
make S1Ps) had no effect on known calcium-signaling pathways under the
conditions tested. Endogenous S1P may also participate in the normal
diauxic shift of yeast cultures (8, 16). We have not yet
detected significant Ca2+ signaling events during the
transition to diauxic growth. Therefore, S1P-mediated calcium signaling
represents a novel calcium-signaling pathway in yeast that is, thus
far, potentially activated by undiscovered stimuli.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RI activates sphingosine kinase, resulting in S1P production. Inhibitors of sphingosine kinases block the agonist-stimulated accumulation of S1P
and also suppress the normal mobilization of Ca2+ stored in
the endoplasmic reticulum (2). In permeabilized cells, S1P also
triggers the release of Ca2+ through a mechanism
independent of the known Ca2+ release pathways, suggesting
that S1P activates a novel type of a Ca2+ release channel
(3-5) capable of elevating cytosolic-free Ca2+
concentrations ([Ca2+]c) and stimulating
capacitative Ca2+ entry (CCE) mechanisms. The hypothetical
intracellular S1P receptor and/or Ca2+ release channel has
not yet been identified.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
List of yeast strains used in this study
-Galactosidase Assays--
Cells expressing pKC190 or pDM5
(26, 33) were grown to log phase overnight at 30 °C in SC medium
lacking uracil. Cultures were harvested and resuspended to a final
A600 of 1 in 2 ml of fresh SC lacking uracil or
YPD medium supplemented with sphingosine, NaCl,
-mating factor,
and/or FK506 as noted in the text. Cells were incubated with shaking at
30 °C for 3-4 h before assaying for
-galactosidase activity as
described previously (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Sphingosine 1-phosphate accumulation
specifically stimulates calcium accumulation. A,
wild-type strains and strains lacking either S1P lyase
(dpl1), S1P phosphatase (lcb3) or both were
treated with sphingosine concentrations ranging from 0 to 30 µM. Total cell-associated 45Ca2+
was quantitated after a 3-h incubation period. B, strains
lacking S1P lyase (dpl1), sphingosine kinases (lcb4
lcb5), or both (dpl1 lcb4 lcb5) were treated with
sphingosine and assayed for 45Ca2+ accumulation
as described in A. Results are depicted on separate graphs for clarity.
C, the responses of dpl1 mutants and dpl1
lcb4 lcb5 mutants to D-sphingosine and
L-sphingosine were compared using the
45Ca2+ accumulation assay. Cells were treated
with D-sphingosine (D-Sph),
L-sphingosine (L-Sph), or ethanol
(control) and processed as described above.
View larger version (15K):
[in a new window]
Fig. 2.
Endogenous S1P-related molecules stimulate
Ca2+ accumulation. A, strains lacking S1P
lyase (dpl1), S1P kinases (lcb4 lcb5), or both
were treated with sphingosine, dihydrosphingosine, or phytosphingosine
and assayed for 45Ca2+ accumulation as
described earlier. Calcium accumulation in kinase-deficient strains was
subtracted from those in which the kinases were present to normalize
for kinase-dependent calcium accumulation. B,
wild type (WT) and dpl1 lcb3 double mutant
strains and the corresponding sphingosine kinase-deficient
strains were placed in SC medium containing
45Ca2+, and samples of each were harvested at
6-min intervals for 30 min. Total cell-associated calcium was
determined, and the slopes of each estimated the line used to determine
the rates of calcium accumulation for each strain.
View larger version (14K):
[in a new window]
Fig. 3.
S1P stimulates Ca2+ accumulation
via Cch1p-dependent and Cch1p-independent pathways.
45Ca2+ accumulation assays were performed for
3 h at 30 °C using various concentrations of added sphingosine
(A) or 12.5 µM sphingosine using
dpl1 and dpl1 cch1 mutants (B)
containing or lacking the S1P kinases (Lcb4p and Lcb5p).
View larger version (24K):
[in a new window]
Fig. 4.
S1P accumulation elevates
[Ca2+]c. Aequorin luminescence in
dpl1 mutants and dpl1 lcb4 lcb5 triple mutants
was monitored in cells treated with 12.5 µM sphingosine
in the presence or absence of 10 mM BAPTA (A) or
5 µg/ml cycloheximide (B) and incubated at 30 °C. At
intervals, cultures were vortexed and placed in a luminometer
for quantitation of luminescence. RLU, relative light
units.
View larger version (17K):
[in a new window]
Fig. 5.
S1P accumulation causes
calcineurin-dependent induction of PMC1-lacZ
expression. Various strains carrying the
calcineurin-dependent reporter gene PMC1-lacZ
were grown in SC lacking uracil medium at 30 °C and treated with
varying concentrations of sphingosine (A) or 12.5 µM sphingosine (B) in the presence or absence
of FK506 or BAPTA as indicated. After a 3-h incubation at 30 °C,
-galactosidase activity was assayed in cell lysates. C,
expression of the PMC1-lacZ reporter gene was
determined as described above, except cultures were grown in YPD medium
lacking sphingosine but supplemented with 10 mM
CaCl2 and FK506 as indicated.
-mating factor, triggers Ca2+ influx and
signaling after a time lag similar to that of sphingosine (18). We
found that
-mating factor stimulated Ca2+ influx and
calcineurin-dependent induction of PMC1-lacZ in
lcb4 lcb5 double mutants to the same degree as in wild type
(Fig. 6A) (data not shown).
Thus, the sphingosine kinases (and presumably their products) were not
required for Ca2+ signaling invoked in response to
-mating factor. The second stimulus tested, high salt, also induced
the calcineurin-dependent expression of
FKS2-lacZ to an equal extent in wild type and in lcb4
lcb5 double mutants (Fig. 6B). Next, the acute heat
shock produced by shifting cells grown at 25-39 °C stimulated a
transient elevation of [Ca2+]c in both
dpl1 and dpl1 lcb4 lcb5 mutants as detected by
aequorin luminescence (Fig. 6C). Hypotonic shock produced by diluting cells grown in standard medium with hypotonic medium (36) also
stimulated aequorin luminescence in both wild type and lcb4
lcb5 double mutants (data not shown). Depletion of
Ca2+ from secretory organelles using pmr1
mutants was shown to stimulate Ca2+ accumulation in yeast
(20, 21, 24). However, 45Ca2+ accumulation in
pmr1 lcb4 lcb5 triple mutants was similar to that of
pmr1 mutants (Fig. 6D). Chlorpromazine treatment
stimulates Ca2+ influx and accumulation in wild-type cells
(37) and to an equal degree in lcb4 lcb5 double mutants
(Fig. 6E). Finally, Ca2+ accumulation in
pgm2 mutants stimulated by growth in galactose medium (17)
also occurred in pgm2 lcb4 lcb5 triple mutants (Fig. 6F). In summary, the sphingosine kinases Lcb4p and Lcb5p
were not required for calcium-signaling events triggered by any of the
seven known stimuli. Therefore, it appears that the upstream stimulus
for S1P signaling in yeast defines a novel event in yeast calcium
signaling.
View larger version (30K):
[in a new window]
Fig. 6.
Physiological stimuli evoke calcium signaling
independent of the sphingosine kinases Lcb4p and Lcb5p.
A, wild-type and lcb4 lcb5 mutant strains
expressing PMC1-lacZ were treated with 20 µM
-mating factor (MF) with or without FK506.
-Galactosidase activity was assayed after incubation for 4 h at
30 °C. B, expression of the
calcineurin-dependent FKS2-lacZ reporter gene in
wild type and lcb4 lcb5 mutant strains was measured after a
4-h growth in YPD medium supplemented with 750 mM NaCl and
FK506. C, luminescence of cytoplasmic aequorin expressed in
dpl1 and dpl1 lcb4 lcb5 mutant strains was
measured in the presence or absence of 5 mM BAPTA before
and after shifting parallel cultures to 39 °C. RLU, relative light
units. D, 45Ca2+ accumulation into
wild type and pmr1 mutants and pmr1 lcb4 lcb5
triple mutants was measured during a 4-h growth in YPD medium.
E, 45Ca2+ accumulation into wild
type and lcb4 lcb5 double mutants was measured after 90 min
of treatment with chlorpromazine. F,
45Ca2+ accumulation into pgm2
mutants, pgm2 lcb4 lcb5 triple mutants, and control strains
was measured after a 4 h growth in YPGal medium (yeast
extract/peptone/galactose). All of these conditions evoked calcium
signaling independent of Lcb4p and Lcb5p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank David Bedwell for yeast strains and plasmids and Fujisawa USA, Inc., for the gift of FK506.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Searle Scholars Program/The Chicago Community Trust (to K. W. C.) and National Institutes of Health Grants CA77528 (to J. D. S.), GM41302 (to R. C. D.), and GM53082 (to K. W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-7844; Fax: 410-516-5213; E-mail: kwc@jhunix.hcf.jhu.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010221200
2 E. Muller E. Locke, and K. W. Cunningham, submitted for publication.
3 C. J. Birchwood, J. D. Saba, and K. W. Cunningham, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: S1P, sphingosine 1-phosphate; [Ca2+]c, cytosolic-free Ca2+ concentration; CCE, capacitative calcium entry; SC, synthetic complete; IP3, inositol 1,4,5-trisphosphate; YPD, yeast extract/peptone/dextrose; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Spiegel, S. (1999) J. Leukocyte Biol. 65, 341-344[Abstract] |
2. | Choi, O. H., Kim, J. H., and Kinet, J. P. (1996) Nature 380, 634-636[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Mattie, M.,
Brooker, G.,
and Spiegel, S.
(1994)
J. Biol. Chem.
269,
3181-3188 |
4. |
Ghosh, T. K.,
Bian, J.,
and Gill, D. L.
(1994)
J. Biol. Chem.
269,
22628-22635 |
5. | Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653-1656[Medline] [Order article via Infotrieve] |
6. | Patton, J. L., and Lester, R. L. (1991) J. Bacteriol. 173, 3101-3108[Medline] [Order article via Infotrieve] |
7. |
Nagiec, M. M.,
Skrzypek, M.,
Nagiec, E. E.,
Lester, R. L.,
and Dickson, R. C.
(1998)
J. Biol. Chem.
273,
19437-19442 |
8. | Lanterman, M. M., and Saba, J. D. (1998) Biochem. J. 332, 525-531[Medline] [Order article via Infotrieve] |
9. |
Kim, S.,
Fyrst, H.,
and Saba, J.
(2000)
Genetics
156,
1519-1529 |
10. |
Qie, L.,
Nagiec, M. M.,
Baltisberger, J. A.,
Lester, R. L.,
and Dickson, R. C.
(1997)
J. Biol. Chem.
272,
16110-16117 |
11. |
Mandala, S. M.,
Thornton, R.,
Tu, Z.,
Kurtz, M. B.,
Nickels, J.,
Broach, J.,
Menzeleev, R.,
and Spiegel, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
150-155 |
12. |
Mao, C.,
Wadleigh, M.,
Jenkins, G. M.,
Hannun, Y. A.,
and Obeid, L. M.
(1997)
J. Biol. Chem.
272,
28690-28694 |
13. |
Saba, J. D.,
Nara, F.,
Bielawska, A.,
Garrett, S.,
and Hannun, Y. A.
(1997)
J. Biol. Chem.
272,
26087-26090 |
14. |
Skrzypek, M. S.,
Nagiec, M. M.,
Lester, R. L.,
and Dickson, R. C.
(1999)
J. Bacteriol.
181,
1134-1140 |
15. | Mao, C., Saba, J. D., and Obeid, L. M. (1999) Biochem. J. 342, 667-675[CrossRef][Medline] [Order article via Infotrieve] |
16. | Gottlieb, D., Heideman, W., and Saba, J. D. (1999) Mol. Cell. Biol. Res. Commun. 1, 66-71[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Fu, L.,
Miseta, A.,
Hunton, D.,
Marchase, R. B.,
and Bedwell, D. M.
(2000)
J. Biol. Chem.
275,
5431-5440 |
18. |
Ohsumi, Y.,
and Anraku, Y.
(1985)
J. Biol. Chem.
260,
10482-10486 |
19. |
Mendoza, I.,
Rubio, F.,
Rodriguez-Navarro, A.,
and Pardo, J. M.
(1994)
J. Biol. Chem.
269,
8792-8796 |
20. | Halachmi, D., and Eilam, Y. (1996) FEBS Lett. 392, 194-200[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Strayle, J.,
Pozzan, T.,
and Rudolph, H. K.
(1999)
EMBO J.
18,
4733-4743 |
22. | Paidhungat, M., and Garrett, S. (1997) Mol. Cell. Biol. 17, 6339-6347[Abstract] |
23. | Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon, G., and Sanders, D. (1997) FEBS Lett. 419, 259-262[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Locke, E. G.,
Bonilla, M.,
Liang, L.,
Takita, Y.,
and Cunningham, K. W.
(2000)
Mol. Cell. Biol.
20,
6686-6694 |
25. | Cunningham, K. W., and Fink, G. R. (1994) J. Cell Biol. 124, 351-363[Abstract] |
26. | Cunningham, K. W., and Fink, G. R. (1996) Mol. Cell. Biol. 16, 2226-2237[Abstract] |
27. | Pozos, T. C., Sekler, I., and Cyert, M. S. (1996) Mol. Cell. Biol. 16, 3730-3741[Abstract] |
28. | Antebi, A., and Fink, G. R. (1992) Mol. Biol. Cell 3, 633-654[Abstract] |
29. |
Sorin, A.,
Rosas, G.,
and Rao, R.
(1997)
J. Biol. Chem.
272,
9895-9901 |
30. | Rudolph, H. K., Antebi, A., Fink, G. R., Buckley, C. M., Dorman, T. E., LeVitre, J., Davidow, L. S., Mao, J. I., and Moir, D. T. (1989) Cell 58, 133-145[Medline] [Order article via Infotrieve] |
31. | Nakajima-Shimada, J., Iida, H., Tsuji, F. I., and Anraku, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6878-6882[Abstract] |
32. | Tanida, I., Takita, Y., Hasegawa, A., Ohya, Y., and Anraku, Y. (1996) FEBS Lett. 379, 38-42[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Matheos, D. P.,
Kingsbury, T. J.,
Ahsan, U. S.,
and Cunningham, K. W.
(1997)
Genes Dev.
11,
3445-3458 |
34. | Guarente, L. (1983) Methods Enzymol. 101, 181-191[Medline] [Order article via Infotrieve] |
35. | Dickson, R. C., and Lester, R. L. (1999) Biochim. Biophys. Acta 1426, 347-357[Medline] [Order article via Infotrieve] |
36. |
Batiza, A. F.,
Schulz, T.,
and Masson, P. H.
(1996)
J. Biol. Chem.
271,
23357-23362 |
37. | Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242-248[Medline] [Order article via Infotrieve] |
38. |
Olivera, A.,
Edsall, L.,
Poulton, S.,
Kazlauskas, A.,
and Spiegel, S.
(1999)
FASEB J.
13,
1593-1600 |
39. |
Mathes, C.,
Fleig, A.,
and Penner, R.
(1998)
J. Biol. Chem.
273,
25020-25030 |
40. |
Beeler, T.,
Gable, K.,
Zhao, C.,
and Dunn, T.
(1994)
J. Biol. Chem.
269,
7279-7284 |