Full text of DOI:10.1359/jbmr.2002.17.5.869

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 5, 2002
©
2002 American Society for Bone and Mineral Research
Prostaglandin E Induces Interaction Between hSlo
2
Potassium Channel and Syk Tyrosine Kinase in
Osteosarcoma Cells
1
1
2
ROGER REZZONICO, ANNIE SCHMID-ALLIANA, GEORGES ROMEY,
1
1
1
´
ISABELLE BOURGET-PONZIO, VERONIQUE BREUIL, VIOLETTE BREITTMAYER,
SOPHIE TARTARE-DECKERT, BERNARD ROSSI, and HEIDY SCHMID-ANTOMARCHI
3
1
1
ABSTRACT
Prostaglandins (PGs) are important mediators of bone response to growth factors, hormones, inflammation,
or mechanical strains. In this study, we show that in MG63 osteosarcoma cells, prostaglandin E (PGE )
2
2
2
؉
؉
produces the opening of a large conductance Ca -dependent K channel (BK). This PGE -mediated channel
2
opening induces the recruitment of various tyrosine-phosphorylated proteins on the hSlo ␣-subunit of BK.
Because the C-terminal domain of hSlo encompasses an immunoreceptor tyrosine-based activation motif
(
ITAM), we show that the Syk nonreceptor tyrosine kinase, reported yet to be expressed mainly in hemato-
poietic cells, is expressed also in osteoblastic cells, and recruited on this ITAM after a PGE -induced
2
docking/activation process. We show that Syk/hSlo association is dependent of an upstream Src–related
tyrosine kinase activity, in accord with the classical two-step model described for immune receptors. Finally,
we provide evidence that this Syk/hSlo interaction does not affect the electrical features of BK channels in
osteosarcoma cells. With these data, we would like to suggest the new notion that besides its conductance
function, hSlo channel can behave in bone cells, as a true transduction protein intervening in the bone
remodeling induced by PGE . (J Bone Miner Res 2002;17:869–878)
2
Key words: prostaglandin E , Slo potassium channel, Syk, Src, osteoblast
2
INTRODUCTION
COX-2 mediates increased prostaglandin E
duction in bone in response to various stimuli including
(PGE ) pro-
2 2
ROSTAGLANDINS (PGS) are key mediators of inflamma- parathyroid hormone (PTH), mechanical strain, or inflam-
P
tion with an autocrine/paracrine role in bone metabo- matory cytokines such as tumor necrosis factor (TNF) ␣,
(2–9)
lism. However, the role of PGs in bone appears complex, interleukin (IL) 1␤, IL-6, IL-11, and IL-17.
because they have been shown to have both stimulatory and
PGE has been studied extensively in the past and like
2
(1)
inhibitory effects on bone formation and bone resorption.
many resorptive cytokines exhibits pleiotropic effects on
Cyclo-oxygenases (COXs) catalyze the first step in the
conversion of arachidonic acid (AA) to prostanoids, includ-
ing PGs. Recent studies suggest that the inducible isoform
bone metabolism. PGE acts directly on the physiology of
2
osteoblasts with indirect consequences on the osteoclastic
bone resorption. Recently, PGE has been shown to stimu-
2
late the expression of the osteoclast differentiation factor
The authors have no conflict of interest.
(ODF)/receptor activator of nuclear factor ␬B (NF-␬B) li-
1
INSERM U364, IFR 50, Facult e´ de M e´ decine, 06107 Nice Cedex 02, France.
CNRS UPR 411, IPMC, 660 route des Lucioles 06560, Valbonne, France.
INSERM U145, IFR 50, Facult e´ de M e´ decine, 06107 Nice Cedex 02, France.
2
3
8
69

8
70
REZZONICO ET AL.
gand (RANKL)/osteoprotegerin ligand (OPGL)/TNF- Piceatannol and 4-amino-5-(4-chlorophenyl)-7-(t. butyl)
(10)
related activation cytokine (TRANCE) in osteoblasts.
pyrazolo [3,4-d] pyrimidine (PP2) were from Calbiochem
Moreover, PGE can inhibit bone resorption by a direct (La Jolla, CA, USA) and all other chemicals were from
2
(11)
action on mature osteoclasts.
Sigma (St. Louis, MO, USA).
In osteoblasts, PGE , PTH, and mechanical loading stim-
2
ulate an increase in intracellular calcium, likely via the
production of inositol triphosphate, diacylglycerol, and cy-
Cell lines, cell transfection, and preparation
of cell lysates
(12–14)
clic adenosine monophosphate (cAMP).
This increase
2
ϩ
in intracellular Ca has been shown to trigger the opening
The human MG63 osteosarcoma cell line was obtained
2
ϩ
of Ca -dependent K channels of high conductance from American Type Culture Collection (ATCC; Rockville,
(
15,16)
(
BK).
eling, that this type of channel also has been shown to mented with 10% (vol/vol) heat-inactivated fetal calf serum
exhibit mechanosensitive properties in osteoblasts and (FCS) (Biowhittaker, Gagny, France),
mM of
L-glutamine, 50 IU/ml of penicillin, and 50 ␮g/ml of strep-
It is noteworthy, in the context of bone remod- MD, USA). Cells were cultured in RPMI 1640 supple-
2
(
16,17)
odontoblasts.
ϩ
In contrast to other voltage-dependent K channels, the tomycin in 5% CO -air–humidified atmosphere at 37°C.
2
pore-forming ␣-subunit of BK channels is encoded by a Cells were starved 16 h in RPMI 1640 before exposure to
(
18,19)
single gene slo (slowpoke),
that undergoes extensive, various effectors. The human CAL-72 osteosarcoma cell
(20,21)
(41)
hormonally regulated, alternative RNA splicing.
These line that we have previously characterized
was main-
splice variants give rise to channels with different functional tained like MG63 cells. CAL-72/Syk stable transfectants
properties, including calcium sensitivity, single-channel were established using the Transfast Promega kit (Promega,
6
conductance, and regulation by intracellular signaling Madison, WI, USA). Briefly, 10 cells were plated onto
(
22–25)
pathways.
In addition, association of the Slo ␣-subunit petri dishes 16 h before transfection with pEF-Bos/Syk (8
with accessory subunits modulates channel characteristics ␮g) ϩ pcDNA3 (2 ␮g) according to the manufacturer’s
(26–29)
or their cellular distribution.
Large-conductance protocol. Cells were cultured for 48 h and then selected in
calcium-activated potassium channels also are subject to RPMI complete medium containing 1 mg/ml of G418 (Life
(
26,30–32)
(33,34)
modulation by protein kinases,
phosphatases,
Technologies, Paisley, UK). Clones were isolated and
screened by Western blotting for Syk expression.
COS cells were maintained in Dulbecco’s modified Ea-
(
35–37)
and other signaling proteins.
In this study we address the question of the involvement
of BK channels in the triggering and propagation of signal- gle’s medium (DMEM) supplemented with 10% (vol/vol)
ing, via PGE , in a human osteoblastic model. We provide heat-inactivated FCS (Life Technologies), L-glutamine (2
2
2
ϩ
the first evidence that on its Ca -activated form, the hSlo mM), penicillin (100 IU/ml), and streptomycin (100 ␮g/ml)
-subunit of BK channels is phosphorylated by the Src and were transiently transfected by the diethylaminoethyl
␣
kinases on an immunoreceptor tyrosine-based activation (DEAE)-dextran method with complementary DNA
6
motif (ITAM) consensus sequence that allows the subse- (cDNA) plasmids. Briefly, cells (10 cells) were plated onto
quent recruitment and activation of Syk, a tyrosine kinase petri dishes 16 h before transfection. Cells were washed
known to play a crucial role in the activation of various cell twice with serum-free DMEM and incubated for 3 h at 37°C
(
38–40)
types.
This leads to the concept that BK channels can in the transfection medium (0.8 ␮g/ml of DNA, 0.1 ␮M of
behave not only as ion pores but also as true transducing chloroquine, and 500 ␮g/ml of DEAE-dextran in DMEM
proteins. This newly identified role of BK channels is likely supplemented with 10% [vol/vol] NU serum [Pharmingen,
to have important implications in the understanding of the San Diego, CA, USA]). Then, cells were treated with 10%
early biochemical events intervening in the activation of dimethyl sulfoxide in phosphate-buffered saline (PBS) for 2
osteoblasts.
minutes at room temperature and cultured in DMEM sup-
plemented with 10% (vol/vol) FCS for 48 h.
Stimulated MG63 cells and transfected CAL-72 or COS
cells were washed twice with PBS and harvested by lysis in
ice-cold 1% NP-40 buffer containing 150 mM of NaCl, 0.8
MATERIALS AND METHODS
Antibodies and chemicals
mM of MgCl , 5 mM of EGTA, 1 mM of phenylmethyl-
2
The antibody (Ab) to hSlo was generated by immunizing sulfonyl fluoride (PMSF), 15 ␮g/ml of leupeptin, 1 ␮M of
rabbits with a glutathione S-transferase (GST) fusion pro- pepstatin, 1 mM of Na VO , and 50 mM of HEPES at pH
3
4
tein containing the hSlo amino sequence from residue 805– 7.5 (lysis buffer). The crude lysates were centrifuged at
75 (GST-hSlo). Specific Ab’s to hSlo were purified first 18,000g for 20 minutes at 4°C; the supernatants (lysates)
9
through an affinity column containing the GST protein to were removed and the protein concentration was assayed
eliminate anti-GST Ab’s, followed by a second affinity using the Lowry method (Bio-Rad Laboratories, Hercules,
column containing the GST-hSlo protein. Ab’s to hSlo CA, USA).
ultimately were eluted by an acid shock (0.1 M of HCl/
glycine at pH 2.6). The antiphosphotyrosine (anti-Ptyr;
Plasmids, DNA constructs, and mutagenesis
4
G10), the anti-cSrc (GD11), and the anti-Syk (4D10.1)
monoclonal Ab’s (mAb’s) were from Upstate Biotechnol-
Bluescript II KS ϩ plasmid containing hSlo cDNA, al-
ogy (Lake Placid, NY, USA). The anti-c-myc mAb 9E10 ternative splice exon free, (Genebank access number
was from Roche Diagnostics, Indianapolis, IN, USA). U13913) was provided by Dr. Steven Dworetzky (Walling-

؉
PGE2 RECRUITS Syk ON ITAM OF hSlo K CHANNEL
871
ford, CO, USA). For a more efficient protein expression, Costa-Mesa, CA, USA) for 15 minutes at 30°C. Reactions
hSlo cDNA was subcloned into pRcCMV plasmid in the were stopped by the addition of L a¨ emmli sample buffer,
HindIII and NotI sites. hSlo mutants were generated by boiled at 95°C for 5 minutes, and analyzed by sodium
site-directed mutagenesis of double-stranded DNA using dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-
the Gene Editor system kit (Promega). Tyrosine 1061, ty- PAGE) and autoradiography.
rosine 1072, or both were replaced by phenylalanine resi-
dues (mutants hSlo F1061, hSlo F1072, and hSlo F1061–1072
For immunoblotting analysis, immunoprecipitates were
dissolved in SDS sample buffer, boiled at 95°C for 5 min-
,
respectively). All mutations were verified by DNA sequenc- utes, and directly resolved by 10% SDS-PAGE.
ing analysis. The pSGT-cSrc plasmid was a kind gift of Dr
S. Roche (Montpellier, France). The pEF-Bos expression
vector containing the full-length cDNA coding for human Immunoblotting
Syk was a kind gift from Dr. V. Imbert (Nice, France). The
porcine Myc epitope–tagged Syk expression vector was
SDS-PAGE resolved samples were transferred onto
Immobilon-P membranes (Millipore, Bedford, MA, USA)
generated by cloning the full-length Syk cDNA from PM-
(43)
(42)
as detailed previously.
The immunoblots were incubated
Syk in the EcoRI and XhoI sites of the pCS3 vector.
overnight at 4°C with rabbit anti-hSlo polyclonal Ab, anti-
Src, anti-Syk, anti-c-myc, or anti-Ptyr mAb’s. After, three
washes with 10 mM of Tris at pH 7.4, 150 mM of NaCl, and
Electrophysiology
1
% NP-40, blots were incubated with secondary horse rad-
Three types of recording modes were used: cell-attached
and excised inside-out and outside-out patches. For outside-
out patch recordings, the pipette solution contained 140 mM
ish peroxidase (HRP)–conjugated goat anti-rabbit (GAR) or
HRP-conjugated rabbit anti-mouse (RAM) Ab’s (Dako,
Copenhagen, Denmark) and Ab-bound proteins were de-
tected by the Amersham enhanced chemiluminescence
of KCl, 3 mM of MgCl , and 10 mM of HEPES, adjusted at
2
2
ϩ
pH 7.3, with KOH. The free Ca concentration was ad-
(
ECL) system. In some experiments, autoradiographs were
justed to 1 ␮M (5 mM of EGTA and 4.7 mM of CaCl ). The
2
ϩ
quantified by densitometric scanning using a laser densi-
tometer equipped with ImageQuant software (Molecular
Dynamics, Sunnyvale, CA, USA).
external solution was either a 5-mM K solution containing
1
40 mM of NaCl, 5 mM of KCl, 3 mM of MgCl , 1 mM of
2
CaCl , 5 mM of glucose, and 10 mM of HEPES, adjusted to
2
ϩ
pH 7.4, with NaOH or a K -rich solution that contained 140
mM of KCl instead of 140 mM of NaCl. For cell-attached
and inside-out configurations, the pipette solution was the
RESULTS
ϩ
5
1
-mM K external solution and the bath solution was the
40-mM K external solution.
ϩ
MG63 cells express a PGE -responsive Ca-activated K
2
The cell-surrounding medium was changed rapidly by a
local microperfusion system consisting of a series of stain-
conductance of the BK type
less tubes (100-␮m inner diameter) connected to different
test solutions and capped by a tapered plastic tube (500-␮m osteosarcoma cells revealed the presence of a Ca
tip inner diameter), flowing at a constant rate of about 0.2 sensitive high conductance (Fig. 1A). Ca sensitivity was
ml/minute. The fluid was maintained at a constant level by assessed by exposing inside-out patches to various internal
direct suction.
The experiments were done at room temperature. Data brief in the absence of Ca , while they were frequent and
were recorded using an RK400 path-clamp amplifier (Bio- maintained in 1 ␮M of Ca . Current-voltage relationships
Logic, Claix, France). Single channel currents were moni- obtained from an outside-out patch configuration in physi-
tored and stored continuously using a DAT recorder. Data ological (5 mM of K ) and symmetrical (140 mM) K
were replayed, sampled, and analyzed using pClamp soft- gradients showed reversal potentials of Ϫ75 mV and 0 mV,
ware (Axon Instruments, Union City, CA, USA).
Patch-clamp experiments performed on MG63 human
2
ϩ
-
2
ϩ
2
ϩ
Ca concentrations. Channel openings were both rare and
ϩ
2
2
ϩ
ϩ
ϩ
ϩ
respectively, in accord with a K selectivity for this channel
Fig. 1B). The calculated unitary conductance values for
(
this channel were 95 pS (in 5 mM) and 235 pS (in 140 mM).
Immunoprecipitation and kinase assay
Addition of PGE (10 nM) in a recording configuration of
2
Cell lysates (0.5–1 mg of proteins) were precleared with silent cell-attached patches held at 0 mV induced a frequent
ϩ
rabbit nonimmune serum prebound to protein A–Sepharose and sustained activation of large conductance K channels
(Amersham Pharmacia Biotech, Inc., Piscataway, NJ, after a delay of a few seconds (Fig. 1D). Altogether these
USA). Then hSlo was immunoprecipitated with 1 ␮g of data support the idea that in MG63 cells, PGE induces the
2
2
ϩ
ϩ
polyclonal anti-hSlo, followed by incubation with protein opening of a Ca -sensitive K channel of BK (big con-
A–Sepharose for 1 h. Immunopellets were washed twice ductance) type. Interestingly, in this cell type, the Ca
with lysis buffer and twice with kinase buffer (10 mM of dependent K channel was insensitive to classical blockers
MgCl , 10 mM of MnCl , 1 mM of paranitrophenylphos- of BK channels such as charybdotoxin and iberiotoxin, as
2
ϩ
-
ϩ
2
2
phate, and 50 mM of HEPES, pH 7.4).
shown in Fig. 1C. However, it was totally blocked by
For in vitro kinase assay, immunoprecipitates were incu- application of 1 mM of tetraethyl ammonium (TEA), in
bated in 50 ␮l of kinase buffer containing 10 ␮Ci of accord with previous data on the sensitivity of BK channels
3
2
(44,45)
[
␥- P]adenosine triphosphate (ATP; 370 MBq/ml; ICN, toward this drug.

8
72
REZZONICO ET AL.
FIG. 2. PGE2 induces the recruitment of tyrosine phosphorylated
proteins on hSlo in MG63 cells. (A) Immunodetection of hSlo
␣-subunit of BK channel in MG63 osteosarcoma cells. Whole lysates
and anti-hSlo or anti-GST immunoprecipitates prepared from MG63
cells were subjected to immunoblot analysis with an anti-hSlo Ab as
described in the Materials and Method section. (B) Coimmunoprecipi-
tation of hSlo channel with tyrosine phosphorylated proteins under
PGE2 stimulation. MG63 cells were either untreated or treated with
PGE2 (10 nM) for 5 minutes or 10 minutes. Then, cell lysates were
prepared and subjected to immunoprecipitation using an anti-hSlo Ab.
Tyrosine phosphorylated proteins coimmunoprecipitated with hSlo
channels were analyzed by Western blotting with an anti-Ptyr Ab. Left
and right panels correspond to different times of ECL signal detection.
of hSlo, was both specifically immunoprecipitated and re-
vealed by Western blotting using these Ab’s.
ϩ
FIG. 1. Functionnal expression of high-conductance K channel
BK) in MG63 osteosarcoma cells. (A) Calcium sensitivity of the BK
channel recorded in the inside-out configuration at ϩ50 mV. Replacing
(
2
ϩ
the intracellular solution containing 1 ␮M of free Ca with one hSlo constitutively interacts with Src and recruits Syk
2
ϩ
without Ca
reversibly blocks channel activities. (B) Successive
traces from an outside-out patch containing a BK channel. I-V rela-
on PGE stimulation in MG63 cells
2
tionships obtained with voltage ramp (200 mV/s) between Ϫ80 and
PGE has been reported to increase the cAMP/protein
2
ϩ
ϩ100 mV. Bath solution was either the external 5 mM of K solution kinase A (PKA) and phospholipase C (PLC) pathways
ϩ
ϩ
(
upper traces) or the K -rich (140 mM of K ) external solution (lower
resulting in the phosphorylation of various substrates. To
gain information on the mechanisms through which modu-
lation of the BK channel activity occurs, we analyzed, by
Western blotting, the pattern of phosphotyrosine proteins
2
ϩ
traces); internal free Ca concentration was adjusted to 1 ␮M. Dotted
lines were drawn through the open- and closed-currents levels. Unitary
ϩ
conductances in low and high K external concentration were esti-
mated at 95 pS and 235 pS, respectively. (C) Pharmacologic properties
of the BK channel. In a typical experiment in the outside-out config-
uration, neither charybdotoxin (ChTx; 100 nM) nor iberiotoxin (IbTx;
associated with hSlo/BK channels in response to PGE . To
2
this end, MG63 cells were exposed to PGE for various
2
periods of time before being lysed. Thereafter, hSlo
2
0 nM) blocks channel activity. Tetraethylammonium (TEA; 1 mM)
reversibly inhibits the channel activity (holding potential, ϩ50 mV). ␣-subunit of BK channels was immunoprecipitated with
(
D) Activation of BK channels by PGE2 (10 nM) in a cell-attached specific polyclonal Ab’s and immunopellets were subjected
ϩ
patch with a low basal activity (bath solution, 140 mM of K -external to electrophoresis, transferred onto a nylon membrane, and
solution; pipette solution, 5 mM of K external solution; holding
potential, 0 mV). Application of PGE is indicated by a horizontal bar.
ϩ
Western blotted with Ab’s to phosphotyrosine. As shown in
Fig. 2B several proteins interacted with hSlo in response to
2
PGE in a time-dependent manner, exhibiting various levels
2
of phosphorylation, with molecular mass ranging from 60 to
Given that BK conductances have been reported to de- 120 kDa.
(
22,24,46)
pend on the expression of the hSlo protein,
we
Recently, A direct interaction of Drosophila and mouse
(30,47)
produced polyclonal Ab’s to the recombinant C terminus Slo with the Src tyrosine kinase has been reported.
part of hSlo to assess the expression of this protein in MG63 This prompted us to investigate whether this kinase also
cells. As shown in Fig. 2A, a protein migrating around interacted with hSlo in our system. To address this question,
1
10–120 kDa, consistent with the expected molecular mass lysates from MG63 cells preexposed to PGE for various
2

؉
PGE2 RECRUITS Syk ON ITAM OF hSlo K CHANNEL
873
FIG. 4. An ITAM is present in the cytoplasmic tail of hSlo. The
minimal consensus ITAM sequence (underlined) consists of two
YxxL/I repeats (one letter code of amino acids, where “x” stems for any
residue), 6–8 residues apart.
periods of time were subjected to coimmunoprecipitation
experiments. As shown in Fig. 3A, Src was constitutively
associated with hSlo and was not recruited further on PGE2
treatment. Consistently, we showed that in CAL-72 osteo-
sarcoma cells, which expressed high endogenous level of
hSlo, the overexpression of Src resulted in the formation of
a constitutive Src-hSlo complex (Fig. 3B). Along the same
line, when Src and Slo were coexpressed in COS cells, we
did observe the same kind of association (Fig. 3B). Based
on the observation that PGE increased the extent of ty-
2
rosine phosphorylation of a band traveling with an apparent
M of 120 (Fig. 2B), we sought to determine whether this
r
band corresponded to a Src-dependent phosphorylated form
of hSlo. Indeed, we found that the level of tyrosine phos-
phorylation of the 120-kDa band, immunoprecipitated by
anti-hSlo Ab’s, was increased by exposure to PGE whereas
2
(48)
PP2, a potent inhibitor of Src family tyrosine kinases,
abrogated this phosphorylation (Fig. 3C, left panel). The
Src-dependent phosphorylation of hSlo was confirmed by
the dramatic decrease induced by PP2 on the antiphospho-
tyrosine immunoprecipitable fraction of hSlo (Fig. 3C, righϩt
panel). Contrary to numerous reports on modulation of K
(32)
channel activities by tyrosine kinases,
we found that the
Src-dependent phosphorylation of hSlo had no significant
effect on the electrical features of BK channels in MG63
cells when stimulated either by PGE2 (Fig. 3D) or by
depolarization (Fig. 3E).
The analysis of the hSlo sequence reveals the presence
of an ITAM motif in the C terminus tail of the protein
Fig. 4). This consensus sequence has been proven to be
essential for the docking of Syk tyrosine kinase on im-
(
FIG. 3. cSrc is associated with hSlo and participates in its tyrosine
and reprobed with anti-hSlo. (C) Effect of PGE2 and PP2 on hSlo
tyrosine phosphorylation in MG63 cells. Cells were preincubated (or
phosphorylation on PGE -stimulated MG63 cells. (A) Effect of PGE
2
2
on Src/hSlo interaction. MG63 cells were treated for the indicated times not) for 30 minutes with the Src-specific inhibitor PP2 (10 ␮M) and
with PGE2 (10 nM). Cell lysates were prepared and immunoprecipi- then stimulated with PGE (10 nM) for 5 minutes. Immunoprecipitation
2
tated with anti-hSlo Ab. Immune complexes were blotted with anti-
cSrc Ab. Membranes were stripped and reprobed with anti-hSlo Ab to
normalize immunoprecipitation efficiency. (B) Reconstitution of Src/
of hSlo or phosphotyrosine-containing proteins were performed and
analyzed by Western blotting with 4G10 or anti-hSlo Ab’s, respec-
tively. Immunoblots were stripped and reprobed with anti-hSlo Ab. (D)
hSlo interaction in CAL-72 and COS transfected cells. CAL-72 osteo- Activation of BK channel by PGE (10 nM) in the absence or presence
2
ϩ
sarcoma cells, which expressed a high level of native hSlo, were of 10 ␮M of PP2 in a cell-attached patch (bath solution, 140 mM of K
ϩ
transiently transfected with control vector or cDNA coding for cSrc.
external solution; pipette solution, 5 mM of K external solution;
COS cells were transfected with cDNA coding for hSlo and Src, alone holding potential, 0 mV). Application of PGE is indicated by a horizontal
2
or together. After 48 h, cells were lysed and subjected to immunopre-
bar. (E) BK channel activity was measured in cell-attached patches, hold-
cipitation with anti-hSlo Ab, and immunoprecipitates were analyzed by ing potential near ϩ50 mV, in the absence or presence of 10 ␮M of PP2.
ϩ
2ϩ
immunoblotting with an anti-cSrc Ab. Then, membranes were stripped
Bath and pipette solutions were 140 mM of Na and 2 mM of Ca .

8
74
REZZONICO ET AL.
FIG. 6. A Src-like tyrosine kinase is necessary for Syk/hSlo interac-
tion. (A) MG63 cells were preincubated or not preincubated for 30
minutes with PP2 (10 ␮M) before stimulation in the presence of PGE2
for 5 minutes. Then, cell lysates were immunoprecipitated with anti-
hSlo Ab and an in vitro kinase assay was performed as described in the
Materials and Methods section. (B) Cells were processed as in A and
hSlo immunoprecipitates were blotted with anti-Syk Ab. The blot was
reprobed with anti-hSlo as a loading control.
FIG. 5. Syk tyrosine kinase interacts with hSlo. (A) PGE stimulates anti-hSlo immunoprecipitates and, reciprocally, the pres-
2
Syk/hSlo interaction in MG63 cells. Cells were treated with 10 nM of
PGE2 for the indicated times and lysed. Then, cell lysates were sub-
jected to immunoprecipitation using anti-hSlo Ab and the immune
ence of hSlo in anti-Syk immunopellets (Fig. 5B).
An Src-like tyrosine kinase activity is necessary for the
complexes were analyzed by Western blotting for the presence of Syk.
The same membrane was stripped and reprobed for hSlo. The diagram docking of Syk on hSlo ITAM
shows the densitometric scanning quantification of Syk signals normal-
Syk recruitment on ITAM of immunoreceptors has been
shown to require prior phosphorylation of the two tyrosine
ized to hSlo. (B) Reconstitution of Syk/hSlo interaction in CAL-72–
transfected cells. CAL-72 cells stably expressing Syk were established
(50)
as described in the Materials and Methods section. The upper panel residues of this motif by Src-related tyrosine kinases.
In
shows the Western blot analysis of native hSlo and exogenous Syk this regard, it is of particular interest that the Src inhibitor
expression in whole lysates from parental and Syk-transfected CAL-72 PP2 altered the level of PGE -induced hSlo/Syk association,
2
cells, respectively. In the lower panel, cell lysates from both cell types
were immunoprecipitated with anti-hSlo or anti-Syk Ab’s and analyzed
by successive anti-hSlo or anti-Syk hybridizations.
as evidenced by kinase assay (Fig. 6A) or anti-Syk immu-
noblotting experiments (Fig. 6B) performed on anti-hSlo
immunoprecipitates. To better delineate the mode of inter-
action of Syk on the hSlo sequence, we carried out separate
or combined site-directed mutagenesis of the two tyrosine
Furthermore, the major tyrosine phos- residues located within the C-terminus ITAM of hSlo. COS
(
49)
mune receptors.
phorylated band associated with hSlo exhibited an appar- cells were transfected with both hSlo and myc-tagged Syk
ent M of 70–72. Taken together, these data prompt us to constructions. In this model, ectopic high expression of
r
investigate the possibility that this protein corresponded Syk-myc and hSlo resulted in a constitutive association
Syk
to p72 . In fact, Syk is expressed mainly in immune between the two partners. Data presented in Fig. 7A show
(
50)
cells,
although recent studies have reported also ex- that single substitution of Tyr 1061 or Tyr 1072 for Phe was
pression of this nonreceptor tyrosine kinase in other cell without any effect on hSlo/Syk-myc association. In contrast,
(
51,52)
types.
We observed that Syk was indeed expressed combined mutation of both Tyr 1061 and 1072 residues
in MG63 osteosarcoma cells by Western blotting with markedly diminished the association of Syk with hSlo,
anti-Syk Ab’s. Furthermore, when MG63 cells were ex- showing the importance of phosphorylation of these two
posed to PGE , we detected a time-dependent recruitment residues in the control of Syk/hSlo interaction.
2
of Syk in anti-hSlo immunopellets (Fig. 5A) after kinet-
Futhermore, we showed that substitution of Tyr 1061 and
ics compatible with those observed for the PGE trig- 1072 for Phe was without any effect on the open probability
2
gered association with tyrosine phosphorylated cellular of BK channels when expressed in COS cells (Fig. 7B).
substrates (Fig. 2B). In an attempt to reveal a direct
At this stage, it was of interest to investigate whether the
hSlo/Syk interaction, we made use of CAL-72 cells that intrinsic tyrosine kinase activity of Syk also participated in
do not express Syk while they exhibit high level of hSlo. the control of Syk/hSlo interaction. As shown in Fig. 7C, we
When CAL-72 cells were stably transfected with p72Syk observed, in the same doubly transfected COS cells, that
cDNA, we detected the constitutive presence of Syk in addition of piceatannol, a potent specific inhibitor of Syk

؉
PGE2 RECRUITS Syk ON ITAM OF hSlo K CHANNEL
875
FIG. 8. Syk tyrosine kinase does not affect BK channel activity in
osteosarcoma cells. (A) Representative traces of BK channel activity
recorded in the outside-out configuration at ϩ50 mV from parental or
stably Syk-transfected CAL-72 cells. Bath solution was the external
ϩ
ϩ
5
-mM K solution; the 140-mM K pipette solution contained 1 ␮M
2
ϩ
of Ca . (B) CAL-72/Syk cells were preincubated or not preincubated
with piceatannol (5 ␮g/ml) for 1 h and BK channel activity recorded in
the cell-attached configuration at 0 mV. Bath and pipette solutions were
ϩ
the 5-mM K external solution.
modulated through Syk/hSlo interaction in osteosarcoma
cells.
DISCUSSION
FIG. 7. hSlo ITAM sequence is required to permit Syk/hSlo interac-
tion. (A) Role of hSlo ITAM motif in the interaction with Syk. hSlo
ITAM mutations were generated by site-directed mutagenesis as de-
scribed in the Materials and Methods section. COS cells were tran-
siently transfected with vectors coding for the indicated cDNA (Syk-
PGE has been shown to play an important role in the
2
orchestration of bone remodeling, especially through its
activating effect on osteoblasts. Interestingly, PGE2 has
been shown to increase the intracellular calcium concentra-
2
ϩ
myc corresponding to a myc-tagged form of Syk). Cell lysates were tion and, consequently, the opening of Ca -dependent po-
(15)
2ϩ
subjected to anti-hSlo immunoprecipitation and successively immuno- tassium channels. In this study, we focused on the Ca
-
blotted with anti-Myc and anti-hSlo Ab’s. (B) Representative traces of dependent large conductance potassium channel, because of
BK channel activity in the outside-out configuration at ϩ50 mV in
its expression in osteoblasts, and its ability to open in
COS cells transiently transfected with vectors coding for hSlo wt or
response to PGE . We first verified that the product of the
2
ϩ
mutated hSlo F1061–1072. Bath solution was the external 5-mM K
hslo gene, responsible for the BK conductance, was indeed
expressed in the MG63 osteosarcoma cell line. Exposure of
ϩ
2ϩ
solution; the 140-mM K pipette solution contained 1 ␮M of Ca . (C)
Role of Syk tyrosine kinase activity on Syk/hSlo interaction. COS cells
transiently transfected with the indicated vectors were treated or not
treated with the Syk-specific inhibitor piceatannol (5 ␮g/ml) for 1 h
before being lysed. Cell lysates were immunoprecipitated with anti-
these cells to PGE triggered the opening of a potassium
2
conductance that exhibited all the electrical features and
2
ϩ
ϩ
sensitivity to TEA that hallmark a Ca -dependent K
hSlo Ab and immunoblotted with anti-Myc Ab. Then, the membrane channel of BK type, with the exception that it was insensi-
was stripped and reprobed with the anti-hSlo Ab.
tive toward the two blockers iberiotoxin and charybdotoxin.
This particularity distinguishes it from most of the BK
(45)
channels studied in other tissues.
A possible explanation
(
53)
activity,
was without any effect on Syk recruitment, lies in the fact that BK channels are organized as a multimer
ruling out the possibility that the intrinsic tyrosine kinase complex of ␣-subunits carrying the pore function and
activity of Syk intervened, by itself, in the control of its ␤-regulatory subunits that have been shown recently to
interaction with hSlo. Moreover, Syk overexpression did modulate both the electrical and the pharmacologic features
(54)
not modify the electrical features of BK channels in stably of the channels.
Indeed, we verified that all the
transfected Syk-expressing CAL-72 cells (Fig. 8A). Con- ␤-subunits known (␤1–␤4) were expressed in MG63 cells
versely, Syk inhibition by piceatannol also was without any (data not shown), allowing us to think that the resulting
effect on the aperture of BK channels (Fig. 8B). Taken ␣␤-oligomers mixture may generate channels displaying
together, these data suggest that BK channel activity is not peculiar tissue-specific features.

8
76
REZZONICO ET AL.
(
60)
Syk interacts with an ITAM located in the C terminus
of the hSlo sequence
with Src-like kinases, for its interaction with integrins.
We verified that piceatannol, an inhibitor of Syk activity,
was without any effect on the Syk/hSlo interaction, indicat-
ing that docking of Syk to hSlo did not imply an autocata-
lytic process.
Based on the two-step Src-dependent process described in
immune cells, we verified that Src was constitutively asso-
The large intracytoplasmic C-tail of hSlo is a distinctive
feature among the members of the KCa channel family.
Analysis of this domain revealed that it contains a well-
conserved ITAM sequence, which consists of two
YXX(L/I) cassettes separated by 6–8 amino acids. This was
an unexpected observation, because this kind of motif has
only been reported so far to be part of the sequence of
immune receptors such as the ␨-chain of the T-cell receptor,
the ␣- and ␤-chains of the B-cell receptor, and the Fc␥-
(30)
ciated to hSlo in osteosarcoma cells. Moreover, we found
that PP2, a potent inhibitor of Src-related kinases, abolished
the interaction of Syk with hSlo, suggesting strongly that
phosphorylation of the tyrosine residues of the ITAM motif
by an Src-like kinase was necessary for docking Syk onto
the cytoplasmic domain of hSlo. To further assess the mo-
lecular basis of the hSlo/Syk interaction, each or both of the
tyrosine residues located within the ITAM motif were re-
placed for phenylalanine. It turned out that the two tyrosine
residues needed to be mutated at the same time to signifi-
(49)
receptor of macrophages.
In immune cells, engagement
of these receptors has been shown to activate Src-related
kinases that phosphorylate the two tyrosine residues in the
ITAM sequence. This phosphorylation process confers to
ITAM the ability to interact with the two tandem Src ho-
(55)
mology 2 domains of Syk or Zap 70.
Syk is a protein
cantly alter, but not abrogate, the hSlo/Syk association. In
tyrosine kinase (PTK) that is widely expressed in hemato-
poietic cells, whereas its congener Zap 70 is exclusively
(49)
contrast with studies carried out in immune cells,
but in
accord with data performed in platelets aiming to study the
(56)
expressed in T lymphocytes and natural killer (NK) cells.
(60)
interaction of Syk with the ␣ ␤ -integrin,
these data
IIb
3
Both of them are involved in the coupling of activated
immunoreceptors to downstream signaling events in relation
suggest that other domains of the C terminus of hSlo might
participate in the interaction with Syk.
Recently, a large body of evidence has been accumulated,
showing that serine/threonine kinases as well as tyrosine
kinases could regulate the open probability of ion chan-
(57–59)
with proliferation, differentiation, and phagocytosis.
Recently, Syk was shown to be activated on adhesion of
(60)
platelets through the ␣ ␤ -integrin.
In addition to im-
IIb
3
mune cells, Syk expression has been detected in epithelial
(32)
(
51)
(52)
nels.
In this regard, of particular interest is the demon-
cells, fibroblasts, and adipocytes. We verified that this
nonreceptor tyrosine kinase also was expressed largely in
MG63 osteosarcoma cells. Exposure of osteosarcoma cells
(62)
stration, using Syk-deficient DT40B mutants,
that B cell
2
ϩ
receptor (BCR)-mediated Ca response requires Syk acti-
vation. Thus, the question raised is to know whether, in our
to PGE induced the recruitment on hSlo of a panel of
2
2ϩ
model, Syk could exert a negative feedback on the Ca
-
tyrosine phosphorylated proteins, including a protein with
induced opening of hSlo channels. We found that inhibition
of Syk by piceatannol had no effect on BK channel activity
in osteosarcoma cells. Moreover, in transfected CAL-72/
Syk cells stably expressing a high level of Syk, the perma-
nent association of hSlo with Syk failed to modify the
electrical features of BK channels.
Taking our data together, we would like to bring forward
the new concept that besides their ion conductance function,
hSlo channels also can be regarded as true transducing
proteins that, in their open state, can activate a Src-related/
Syk PTK cascade, leading to the phosphorylation of a series
of cellular substrates. Identification of those substrates and
elucidation of the mode of activation of the Src activity will
an apparent M of 72, which corresponds to Syk.
r
Potassium channels have been shown to be targets for
(32)
various protein kinases.
Nevertheless, the effect of these
phosphorylations in terms of open probability is still a
matter of debate. Some potassium channels are intimately
associated with their kinases. It is the case of the voltage-
dependent delayed rectifier potassium channel (Kv1.3),
which was reported to interact with the SH3 domain of
(
61)
Src.
More recently, Drosophila and mouse Slo channels
have been shown to interact constitutively with c-Src and
(30,47)
the catalytic subunit of PKA.
In this context, it was
tempting to explore the exciting hypothesis that the state of
hSlo opening could control its interaction with tyrosine
kinases. Unlike the Src-related members, the Syk/Zap 70
family is devoid of prenylation sites, destining it to a cyto-
plasmic location. In MG63 cells, we observed a slight
constitutive association of Syk with hSlo, which was in-
help in understanding the means by which PGE controls (in
2
osteoblastic cells) the expression of genes important for
bone homeostasis. In this context, BK channels might ap-
pear as important targets for the development of new drugs
aimed at modulating bone remodeling.
creased markedly in response to PGE , suggesting that the
2
potassium channel needs to adopt an open conformation to
interact with Syk.
ACKNOWLEDGMENTS
Recruitment of Syk on hSlo ITAM requires activation
of a Src-related kinase
The authors are indebted to E. Van Obberghen for critical
reading of this study. This work was supported by the
Recruitment of Syk to immune receptors has been shown Institut National de la Sant e´ et de la Recherche M e´ dicale
to be strictly dependent on phosphorylation of ITAM by (INSERM) and the Association pour la Recherche sur le
Src-related kinases. Nevertheless, intrinsic Syk kinase ac- Cancer (ARC; grant 5417). Roger Rezzonico was a recipi-
tivity also has been shown to be required, in conjunction ent of a fellowship from ARC.

؉
PGE2 RECRUITS Syk ON ITAM OF hSlo K CHANNEL
877
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