Laboratory of Physiology, Catholic University of Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
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ABSTRACT |
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We used the whole cell
patch-clamp technique in calf pulmonary endothelial (CPAE) cells to
investigate the effect of wild-type and mutant c-Src tyrosine kinase on
ICl,swell, the swelling-induced Cl
current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect ICl,swell. However, transfection of c-Src
with a Ser3Cys mutation that introduces a dual acylation
signal and targets c-Src to lipid rafts and caveolae strongly repressed
hypotonicity-induced ICl,swell in CPAE cells.
Kinase activity was dispensable for the inhibition of
ICl,swell, since kinase-deficient c-Src
Ser3Cys either with an inactivating point mutation in the
kinase domain or with the entire kinase domain deleted still suppressed
VRAC activity. Again, the Ser3Cys mutation was required to
obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the
inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated
inhibition of VRAC requires compartmentalization of c-Src to caveolae
and that the Src homology domains 2 and/or 3 are necessary and
sufficient for inhibition.
anion channel; caveola; tyrosine kinase; cell volume; volume-regulated anion channel
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INTRODUCTION |
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IN MOST MAMMALIAN CELL
TYPES the primary response to cell swelling includes the
activation of a Cl current through volume-regulated anion
channels (VRACs). This outwardly rectifying current, termed
ICl,swell for swelling-activated Cl
current, is not only involved in cell volume
regulation but also participates in electrogenesis, the control of the
driving force for Ca2+ influx, and more speculatively in
the regulation of intracellular pH and cell proliferation (for reviews,
see Refs. 18, 22, and 31).
A major question with respect to VRAC is how cellular volume expansion
causes channel activation. In calf pulmonary artery endothelial cells
(CPAE), VRAC activation seems to depend on one or more tyrosine
phosphorylation steps, since protein tyrosine kinase (PTK) inhibitors
can inhibit ICl,swell, triggered either by cell
swelling or by alternative isovolumic stimuli such as a decrease in
intracellular ionic strength (i) or intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate)
(GTP
S; see Refs. 19 and 38). Furthermore, protein
tyrosine phosphatase inhibitors potentiate
ICl,swell once it is activated by cell swelling (38). In addition to CPAE cells, evidence for
PTK-dependent activation has also been reported in cardiac myocytes
(30), in human T lymphocytes (14), and human
intestinal 407 cells (33). However, nonhydrolyzable ATP
analogs can sustain VRAC activation in C6 glioma cells
(8) and in N1E115 neuroblastoma cells (3),
indicating that at least in these cell types ATP hydrolysis, and
consequently phosphorylation, is not required for VRAC activation
(3). Currently, it is not clear whether these different
observations have biological relevance (e.g., expression of different
channel isoforms, alternative cell-specific activation pathways) or
whether they are due to methodological or experimental conditions.
Lepple-Wienhues et al. (14) have recently shown that p56lck (or Lck), a lymphocyte-specific PTK belonging to the Src family kinases, mediates the activation of ICl,swell in a human T lymphocyte cell line (14). The vertebrate Src family contains nine PTKs with a conserved domain structure (5, 32). Typically, the catalytic kinase domain is preceded by the proline-binding Src homology domain 3 (SH3) and by the phosphotyrosine- binding Src homology domain 2 (SH2). The kinase domain is followed by a COOH-terminal region containing a conserved, autoinhibitory tyrosine phosphorylation site. Depending on specific stimuli, Src-like kinases adopt either a "closed" inactive or an "open" active conformation. The closed configuration is stabilized by several intramolecular interactions, e.g., between the SH2 domain and the COOH-terminal phosphorylated tyrosine and between the SH3 domain and the linker between the SH2 and the kinase domain (32). Activation is achieved by dephosphorylation of the COOH-terminal tyrosine and/or by binding of proteins containing competing SH2 and/or SH3 domains. Once in the open conformation, Src-like kinases mediate their effect either by tyrosine phosphorylation or by protein-protein interaction via the SH2 and/or SH3 domains.
We have also provided evidence that the VRAC activation cascade is modulated by caveolin-1 (34). Indeed, caveolin-1-deficient Caco-2 cells display little or no ICl,swell upon cell swelling, but transient transfection of caveolin-1 restores the VRAC response (34). Caveolins are 22- to 26-kDa proteins that associate via palmitoyl anchors and an intramembranous hairpin structure with glycosphingolipid- and cholesterol-enriched domains in the plasma membrane (so-called "lipid rafts"; see Ref. 13). The binding of caveolins to lipid rafts generates caveolae, i.e., flask-shaped invaginations of the plasma membrane that have been implicated in endocytosis, signal transduction, and cholesterol transport (1, 16, 23).
Interestingly, Src family kinases can be compartmentalized to lipid
rafts and caveolae (4). Indeed, all vertebrate Src family
kinases, with the exception of c-Src and Blk, contain an NH2-terminal consensus sequence
(Gly2-Cys3) for dual acylation, with
Gly2 being myristoylated and Cys3 palmitoylated
(24). It has been shown for Lck, Fgr, Fyn, and Hck that
dual acylation targets these kinases to rafts and caveolae (25,
27, 36). In contrast, c-Src, which contains a
Gly2-Ser3 sequence at its NH2
terminus, is only monoacylated (myristoylation), resulting in a low
affinity of c-Src for lipid rafts and caveolae (27),
although some groups have reported a small association with caveolae
(29). Importantly, introduction of a Ser3Cys
mutation in c-Src strongly increases its association with rafts and
caveolae (27). This is consistent with the observation that dual acylation strongly enhances the caveolar targeting of other
proteins such as endothelial nitric oxide synthase and
Gi-1 (26, 29). Furthermore, it has been
shown that caveolin-1 binds c-Src via its caveolin-scaffolding domain,
thereby clamping c-Src in the inactive configuration (15).
In view of the link between Src family kinases and caveolae and caveolins and in view of the proposed role of both caveolin and Src family kinases in VRAC activation, we decided to investigate the effect of monoacylated and dually acylated c-Src mutants on ICl,swell in CPAE cells.
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METHODS |
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Cells.
We used single endothelial cells from an established calf pulmonary
artery cell line (CPAE; American Type Culture Collection CCL 209).
Cells were grown in DMEM (Life Technologies, GIBCO) containing 20%
FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. CPAE cells were transiently transfected with the
bicistronic pCINeo/IRES-green fluorescent protein (GFP) vector
(35) containing cDNAs encoding c-Src, c-Src
Ser3Cys, c-Src Tyr527Phe, c-Src
Ser3Cys/Tyr527Phe, c-Src
Ser3Cys/Lys295Arg, c-Src Ser3Cys
kinase, c-Src
kinase, or c-Src Ser3Cys
SH3-SH2.
Cells were directly seeded on gelatin-coated coverslips at 5,000 cells/coverslip 24 h before transfection. Transfection of CPAE
cells was performed using 1 µg plasmid DNA, 2 µl Lipofectamine, and
7 µl "Plus" reagent (LipofectAMINE PLUS; GIBCO-BRL).
cDNA constructs.
A cDNA-encoding chicken c-Src (kindly donated by J. Goris,
Biochemistry, K. U. Leuven, Belgium) was subcloned in the
bicistronic expression vector pCINeo/IRES-GFP (35). Point
mutations were introduced in the c-Src open-reading frame using the
QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer's instructions. The following point
mutations were introduced: Ser3Cys (numbering starts with
the NH2-terminal Met), Lys295Arg, and
Tyr527Phe. c-Src Ser3Cys SH3-SH2 (deletion
of amino acids 81-257) was generated by replacing in frame a
restriction fragment encoding amino acids 1-257 with a PCR
fragment encoding amino acids 1-80, including the
Ser3Cys mutation. c-Src
kinase and c-Src
Ser3Cys
kinase (deletion of amino acids 260-533)
were generated by replacing a restriction fragment encoding amino acids
258-533 with an oligonucleotide containing a stop codon at
position 260 in the c-Src open-reading frame without or with the
Ser3Cys mutation. Mutations were verified by nucleotide
sequencing using the ALF automated sequencer (Amersham Pharmacia
Biotech, Uppsala, Sweden).
Solutions.
The standard extracellular solution was a modified Krebs solution
containing (in mM) 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. The
osmolality of this solution, as measured with a vapor pressure
osmometer (Wescor 5500; Schlag, Gladbach, Germany), was 320 ± 5 mosmol/kgH2O. At the beginning of the patch-clamp
recordings, the Krebs solution was replaced by an isotonic
Cs+ solution, containing (in mM) 105 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, and 90 mannitol, pH 7.4 with NaOH (320 ± 5 mosmol/kgH2O).
The 25% hypotonic solution (HTS) was obtained by omitting 90 mM
mannitol from this solution. The standard pipette solution contained
(in mM) 40 CsCl, 100 cesium aspartate, 1 MgCl2, 1.93 CaCl2, 5 EGTA, 4 Na2ATP, and 10 HEPES, pH 7.2 with CsOH (290 mosmol/kgH2O). The concentration of free
Ca2+ in this solution was buffered at 100 nM. Activation of
VRAC in the absence of an osmotic gradient was achieved by dialyzing
the cells with an isosmotic pipette solution that contained 100 µM GTPS (Sigma) or with an isosmotic pipette solution with reduced ionic strength by substituting 70 mM cesium aspartate with 140 mM sucrose.
Electrophysiological recordings.
Transfected green fluorescent cells were visualized in a patch-clamp
setup, as described previously (35). Currents were monitored with an EPC-7 patch-clamp amplifier (List Electronic, Lambrecht/Pfalz, Germany). Patch electrodes had direct current resistances between 2 and 6 M. An Ag-AgCl wire was used as reference electrode. Whole cell membrane currents were measured using ruptured patches. Currents were sampled at 1-ms intervals and filtered at 1,000 Hz. The following voltage protocol was applied every 15 s from a
holding potential of
25 mV: a step to
80 mV for 0.2 s,
followed by a step to
100 mV for 0.1 s, and a 1.5-s linear voltage ramp to +100 mV. A step protocol was also used, consisting of
1-s voltage steps from a holding potential of
25 mV to potentials ranging from
100 to +100 mV in 20-mV increments. Experiments were
performed at room temperature.
Analysis.
Electrophysiological data were acquired with pCLAMP 5.5 (Axon
Instruments) and were analyzed with WinASCD (G. Droogmans) and Origin
5.1 (Microcal). Time courses of the whole cell current were obtained by
plotting the current at +100 or 100 mV during successive voltage
ramps as a function of potential. Current-voltage relations were
obtained from the currents measured during the linear voltage ramp.
Difference currents (ICl,swell) were calculated by subtracting the basal current under isotonic conditions from the
maximal current during hypotonic stimulation. In the experiments with
intracellular application of GTP
S or reduced ionic strength, the
difference current was calculated by subtracting the basal current
measured at time 0 from either the peak current reached during transient activation with GTP
S or the maximal current at the
plateau phase during intracellular perfusion of a pipette solution with
reduced ionic strength. Pooled data are given as means ± SE from
n cells. Significance between two data sets was tested using
the Student's unpaired t-test. One-way ANOVA was used to
determine statistical differences of three or more data sets.
Differences were considered significant at the level of P < 0.05.
Immunofluorescence. CPAE cells were seeded on gelatin-coated coverslips (7,000 cells/slip) and transfected with pCINeo/IRES-GFP/c-Src S3C. After transfection (48 h), cells were washed three times in PBS and fixed with 3.7% formaldehyde (F-1268; Sigma) in PBS for 10 min at room temperature. After being rinsed with PBS, the cells were permeabilized with 0.2% Triton in PBS for 4 min. The cells were washed with PBS and incubated for 30 min in 10% FCS in PBS. Cells were washed with 1% FCS in PBS and exposed overnight at 4°C to an anti-caveolin-1 polyclonal antibody (diluted 1:400 in PBS containing 1% FCS; Transduction Laboratories C13630) and subsequently for 1 h at room temperature to the secondary tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG (Sigma T-6778; diluted 1:400 in PBS containing 1% FCS). To prevent rapid photobleaching, cells were mounted in Vectashield (J-1000; Vector Laboratories) after washing the coverslips three times in PBS and two times in H2O. Cells were visualized with a Nikon Optiphot-2 microscope using green excitation (510-560 nm) for visualization of TRITC staining and blue excitation (450-490 nm) to identify the GFP signal. Omission of the primary antibody resulted in a very weak or no background staining. As a control, wild-type cells were used.
Western blotting and cell fractionation.
Subcellular distribution of c-myc epitope-tagged c-Src
mutants was studied in Rat-1 cells transiently transfected with
pCINeo/IRES-GFP/c-Src kinase or pCINeo/IRES-GFP/c-Src
Ser3Cys
kinase. Transfected cells (5 × 106) were harvested by scraping in ice-cold 500 µl lysis
buffer (in mM: 25 Tris · HCl, pH 7.2, 50 NaCl, 90 mannitol, and
1 EGTA) supplemented with 1% Triton X-100, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin. The lysate was adjusted to 45% sucrose and subjected to a
sucrose density gradient centrifugation in a Beckman SW50.1 rotor at
250,000 g for 20 h at 4°C. The sucrose gradient was
formed by layering 3 ml of 30% sucrose in lysis buffer and 1 ml of 5%
sucrose in lysis buffer on top of 1 ml lysate-45% sucrose. After
centrifugation, 500-µl fractions were collected from top to bottom
(first 250 µl were discarded), and proteins were precipitated with
10% ice-cold TCA. Pellets were washed with acetone, resuspended in
Laemmli loading buffer, separated by SDS-PAGE (12.5% polyacrylamide), and transferred by semidry electroblotting to a polyvinylidene difluoride membrane (Immobilon; Millipore). Blots were stained with
anti-caveolin-1 monoclonal antibody (C37120; Transduction Laboratories)
or anti-c-myc monoclonal antibody (kind gift of Dr. B. De
Strooper, Center of Human Genetics, K. U. Leuven, Belgium). Immunoreactive bands were visualized with the Vistra enhanced chemifluorescence detection kit (Amersham Pharmacia Biotech) on a Storm
840 Imager (Molecular Dynamics).
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RESULTS |
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c-Src Ser3Cys and c-Src Ser3Cys/Tyr527Phe inhibit ICl,swell in CPAE cells. In a first series of experiments, we investigated the effect of transient expression of c-Src- and c-Src-derived mutants on ICl,swell in CPAE cells. Specifically, we compared monoacylated wild-type c-Src with c-Src mutants that are dually acylated due to a Ser3Cys mutation. As shown by Shenoy-Scaria et al. (27), the Ser3Cys mutation generates a dually acylated c-Src isoform with preferential location to lipid rafts and caveolae (27).
When exposed to a 25% HTS, wild-type CPAE cells generate large, outwardly rectifying chloride currents through VRAC. In wild-type CPAE cells, ICl,swell reached a mean current density of 70.3 ± 7.5 pA/pF at +100 mV (n = 16; Fig. 1A). In CPAE cells overexpressing wild-type c-Src, a 25% HTS evoked a similar increase in ICl,swell, with a mean current density of 55 ± 6.8 pA/pF at +100 mV (n = 21; P > 0.05 when compared with wild-type CPAE cells; Fig. 1A). In contrast, transient transfection of CPAE cells with c-Src Ser3Cys resulted in a significant reduction of ICl,swell: 10.5 ± 1.8 pA/pF at +100 mV (n = 15; Fig. 1A). Subsequently, we analyzed the effect of c-Src Tyr527Phe on VRAC. The Tyr527Phe mutation confers constitutive kinase activity to c-Src (11) because it abolishes the inhibitory interaction between the SH2 domain and the phosphorylated Tyr527 and thus destabilizes the closed inactive conformation (32). ICl,swell density was significantly reduced in CPAE cells transfected with c-Src Tyr527Phe (39.3 ± 5.4 pA/pF at +100 mV; n = 25) compared with nontransfected CPAE cells. However, the inhibitory effect of the Tyr527Phe mutation was less pronounced than that of the Ser3Cys mutation (P < 0.05). Interestingly, the Tyr527Phe-mediated downregulation of ICl,swell was further potentiated by the introduction of a dual acylation signal. Indeed, CPAE cells transiently transfected with c-Src Ser3Cys/Tyr527Phe displayed a very small ICl,swell after a 25% HTS with a mean current density of 18.3 ± 3.5 pA/pF at +100 mV (n = 35; Fig. 1A). This corresponds to a significant reduction compared with nontransfected CPAE cells and CPAE cells transfected with wild-type c-Src or c-Src Tyr527Phe. However, it is not significantly different from ICl,swell in CPAE cells transfected with c-Src Ser3Cys.
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c-Src
Ser3Cys/Tyr527Phe also represses
the isovolumic activation of ICl,swell.
We have recently demonstrated in CPAE cells that VRAC can be triggered
not only by a hypotonic stimulus but also by reducing i
(19, 37) or by intracellular perfusion with GTP
S
(20, 38). We were therefore interested to find out whether
c-Src Ser3Cys/Tyr527Phe also interfered with
the GTP
S- or
i-triggered activation of VRAC, which is
achieved under isovolumic conditions (37, 38). To this
end, we compared the mean current density of
ICl,swell in c-Src
Ser3Cys/Tyr527Phe-transfected CPAE cells during
intracellular perfusion with a pipette solution with reduced
i or containing 100 µM GTP
S. Reduced
i generated a manifest ICl,swell
in wild-type CPAE cells with a mean current density of 44.6 ± 2.3 pA/pF at +100 mV (n = 4), but it was less potent in
eliciting a response in c-Src Ser3Cys/Tyr527Phe-overexpressing CPAE cells
(19.3 ± 6.7 pA/pF at +100 mV; n = 11;
P < 0.05; Fig. 2).
Similarly, intracellular application of 100 µM GTP
S induced a
typical transient activation of VRAC in wild-type CPAE cells (mean
maximal current density of 120.5 ± 26.3 pA/pF at +100 mV;
n = 6), but it hardly evoked a response in CPAE cells
transfected with c-Src S3C/Y527: mean maximal amplitude of 6.7 ± 3.2 pA/pF at +100 mV (n = 9; Fig. 2). Thus c-Src
Ser3Cys/Tyr527Phe represses the swelling
induced and the isovolumic activation of
ICl,swell in transiently transfected CPAE cells,
indicating that the inhibitory effect cannot be attributed to an
indirect action on cell swelling.
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Downregulation of VRAC by c-Src
Ser3Cys is not mediated by the
Src kinase domain but by the SH3-SH2
domains.
To investigate whether the dually acylated c-Src inhibited
ICl,swell by phosphorylating other proteins, we
constructed a kinase-inactive c-Src Ser3Cys mutant by
introducing a Lys295Arg mutation in the ATP-binding site of
the kinase domain (9). In this series of experiments,
ICl,swell reached a mean current density of
100.6 ± 11.6 pA/pF at +100 mV (n = 10; Fig.
3) in wild-type CPAE cells. c-Src
Ser3Cys exerted an inhibitory effect on
ICl,swell (47.0 ± 11.7 pA/pF at +100 mV;
n = 15), albeit somewhat less pronounced than in the previous series of experiments. Importantly, CPAE cells transfected with the kinase-inactive c-Src
Ser3Cys/Lys295Arg generated HTS-induced
membrane currents of similar amplitude (51.2 ± 4.9 pA/pF at +100
mV; n = 19) as CPAE cells expressing c-Src
Ser3Cys (Fig. 3).
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Cofractionation of c-Src
Ser3Cys mutants with caveolin-1-containing
light density fractions.
A consistent finding in the above-described observations is the
critical contribution of the Ser3Cys mutation to the
inhibition of ICl,swell by c-Src mutants (Figs. 1 and 4). Because this mutation introduces a dual acylation signal that
promotes c-Src targeting to lipid rafts and caveolae (27), this strongly suggests that efficient inhibition requires
compartmentalization of c-Src to lipid rafts and/or caveolae. We
therefore verified whether the Ser3Cys mutation indeed
alters the subcellular distribution of c-Src mutants by examining their
cofractionation with caveolin-1 in a sucrose gradient after
solubilization of cells with ice-cold Triton X-100. This procedure
preserves the lipid composition and hence the low buoyant density of
rafts and caveolae (so-called detergent-resistant membranes), which
allows their separation in a sucrose gradient (28). c-Myc
epitope-tagged c-Src kinase and c-Myc epitope-tagged c-Src
Ser3Cys
kinase were transiently expressed in Rat-1
cells, and cell lysates were subjected to sucrose density gradient
centrifugation. Figure 5 shows that the
vast majority of c-Src
kinase stays at the bottom of the gradient
and that only a minor fraction cofractionates with caveolin-1. In
contrast, ~50% of c-Src Ser3Cys
kinase floats up in
the gradient to the low-density, caveolin-1-containing fractions. Thus
the Ser3Cys mutation induces a clear shift in the c-Src
kinase distribution toward the detergent-resistant fraction.
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c-Src Ser3Cys does not
alter the subcellular localization of endogenous caveolin-1 in
CPAE cells.
Because v-Src, a constitutively active oncogenic variant of c-Src,
downregulates the expression and the plasma membrane location of
caveolin-1 and disrupts caveolar structure (12, 21), we investigated whether transient expression of c-Src Ser3Cys
altered the subcellular localization of endogenous caveolin-1. Immunofluorescence microscopy on CPAE cells revealed no significant differences in caveolin-1 expression or subcellular localization between wild-type CPAE cells and CPAE cells transiently transfected with c-Src Ser3Cys (Fig. 6).
Under both conditions, we detected caveolin-1 signals at the cell
periphery (often more concentrated on one edge of the cell). In
addition, some caveolin-1 signals resided from the cell interior, as
can be expected from the dynamic recycling of caveolin-1 between the
plasma membrane and internal organelles (2). The identical
caveolin-1 pattern between control and c-Src Ser3Cys-transfected cells virtually excludes the
possibility that c-Src Ser3Cys inhibits
ICl,swell by altering caveolin-1 expression or
its subcellular distribution. A similar conclusion can also be deduced from Figs. 4 and 5: introduction of the Ser3Cys mutation
significantly increases ICl,swell inhibition
(Fig. 4), yet it does not affect the distribution of caveolin-1 in the sucrose gradient (Fig. 5).
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DISCUSSION |
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In this study, we show that c-Src tyrosine kinase represses VRAC activity in vascular endothelial cells (CPAE) if the c-Src protein is targeted to lipid rafts and caveolae by virtue of a dual acylation signal (provided by the Ser3Cys mutation). Furthermore, the inhibitory effect is mediated by the c-Src SH3-SH2 domains, since deletion of these domains abrogated the inhibition. Vice versa, c-Src mutants lacking the kinase domain still exerted an inhibitory effect on ICl,swell.
The Ser3Cys mutation, which creates a dual acylation signal in the c-Src NH2 terminus, maximalized the inhibition of ICl,swell by c-Src. Because dually acylated proteins are preferentially targeted to lipid rafts and/or caveolae, this observation indicates that these plasma membrane compartments play a crucial role in the expression of VRAC activity. However, it should be stressed that lipid rafts as such (i.e., sphingolipid- and cholesterol-enriched membrane domains) are not sufficient for VRAC activity. Indeed, Triton X-100 solubilization of caveolin-1-deficient Caco-2 cells followed by sucrose density centrifugation revealed the presence of low-density lipid rafts (17). Yet, VRAC could only be efficiently activated in Caco-2 cells after transient transfection with caveolin-1 (34). Together, these data suggest that a crucial component of the VRAC cascade (either the channel itself, an essential regulatory protein, or both) is confined to caveolae, i.e., caveolin-1-containing lipid rafts.
How can we explain the downregulation of VRAC by c-Src targeted to caveolae? One possibility would be that the expression of dually acylated c-Src disrupts caveolar structure and/or composition. However, we could not detect alterations in caveolin-1 expression or in its subcellular distribution in cells transfected with c-Src mutants that clearly exerted an inhibitory effect on VRAC. This observation excludes caveolar disruption as the causative factor. Taking into account that inhibition requires the presence in c-Src of the Src homology domains, which are bona fide protein-protein interaction domains, we envisage two plausible hypotheses. One hypothesis postulates a crucial step in the VRAC activation cascade that is mediated by a caveolar protein containing phosphotyrosine(s) and/or a proline-rich domain. In this scenario, binding of this (as yet unidentified) protein to the c-Src SH2 and/or SH3 domain would result in its sequestration, thereby preventing it from participating in the normal VRAC activation cascade. Such an inhibition by sequestration would resemble the process of "squelching" whereby overexpression of a transcription factor reduces transcriptional activity by titrating out other essential transcription factors (6). The molecular identity of the c-Src interaction partner(s) remains enigmatic, but any candidate should fulfill at least two requirements as follows: 1) it must localize to caveolae and 2) it must be able to interact with SH2 and/or SH3 domains, i.e., it must possess phosphotyrosines, proline-rich domains, or both. Interestingly, Liu et al. (16) have shown that some of the dually acylated Src family kinases, such as Yes, Fyn, Lck, and Lyn, are concentrated in caveolae obtained from lung endothelial cells. In view of the postulated role for tyrosine kinases in VRAC activation in endothelial cells (38) and in view of the requirement for Lck to activate VRAC in lymphocytes (14), it will be interesting to address the role of these dually acylated Src family kinases in the endothelial VRAC cascade.
Alternatively, inhibition of VRAC by caveolar-targeted c-Src can be explained by invoking the caveolar recruitment of an inhibitory protein that normally resides in the cytoplasm. Translocation of this inhibitor to caveolae would depend on its interaction with the SH2 and/or SH3 domain of c-Src. This mechanism bears some resemblance to the Cbp-mediated recruitment of Csk kinase to lipid rafts (10). Cbp is a lipid raft-located phosphoprotein that in its tyrosine-phosphorylated state provides a binding site for Csk, which normally resides in the cytoplasm. After translocation to rafts, Csk phosphorylates the autoinhibitory tyrosine in the COOH terminus of Src family kinases, thereby inactivating the raft-associated Src family kinases (7). Whether overexpression of dually acylated c-Src also induces caveolar translocation of Csk (or of another inhibitory protein) remains to be shown.
We therefore conclude that inhibition of VRAC by c-Src requires compartmentalization of c-Src to caveolae. Furthermore, because SH2 and SH3 are necessary and sufficient to confer inhibition, we suggest that c-Src is incorporated in a caveolar protein-protein interaction network that negatively interferes with the VRAC activation cascade.
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ACKNOWLEDGEMENTS |
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We thank J. Goris (Laboratory of Biochemistry, K. U. Leuven, Belgium) for the gift of c-Src cDNA and B. De Strooper (Center of Human Genetics, K. K. Leuven, Belgium) for the anti-c-myc antibodies. The technical help of J. Prenen, A. Florizoone, M. Crabbé, and H. Van Weijenbergh is greatly appreciated. D. Trouet is a Research Assistant of the Flemish Fund of Scientific Research [Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen].
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FOOTNOTES |
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This research was supported by grants from the Interuniversitaire Attractiepolen (IUAP P4/23) FWO-Vlaanderen (FWO G.0214.99), Geconcerteerde Onderzoeksactie (GOA 99/07), and the `Jean en Alphone Forton' foundation (R7115 B0 to B. Nilius).
Address for reprint requests and other correspondence: J. Eggermont, Laboratory of Physiology, Campus Gasthuisberg, B-3000 Leuven, Belgium (E-mail Jan.Eggermont{at}med.kuleuven.ac.be).
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.
Received 13 April 2000; accepted in final form 12 February 2001.
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