INTRODUCTION

Most cultured fibroblasts respond to the peptide hormone bradykinin with a biphasic elevation of intracellular Ca²⁺ concentration. The initial peak of the Ca²⁺ response is due to Ca²⁺ release from inositol trisphosphate-sensitive stores, whereas the longer duration, plateau phase is due to Ca²⁺ influx from the extracellular medium. Our previous studies have demonstrated that the BK-stimulated¹ Ca²⁺ influx is via a "capacitative" Ca²⁺ pathway (1) similar to the one first described in pancreatic acinar cells by Putney (2). The physiological importance of capacitative Ca²⁺ entry is suggested by the resulting primary immunodeficiency associated with defective T cell proliferation in patients whose lymphocytes have low capacitative Ca²⁺ entry following T cell receptor stimulation (3, 4). Putney hypothesized that the mechanism for opening this type of Ca²⁺ channel involves a signal transduction process in which the "fill state" of the internal Ca²⁺ stores is sensed and a message is sent to open plasma membrane Ca²⁺ channels, if the stores are empty. Subsequent studies from a number of different laboratories have supported the basic concepts of this hypothesis, although the exact mechanism for regulation of these capacitative Ca²⁺ channels, or store-operated channels (SOCs), is still an area of active investigation.

Based upon previous results from our laboratory, we have proposed that a tyrosine kinase activity is involved in relaying the message from the empty internal Ca²⁺ stores to the plasma membrane Ca²⁺ channel (5). This hypothesis is based on our observations that inhibitors of tyrosine kinase activity (such as genistein and tyrphostin) block the plateau phase of the BK-induced Ca²⁺ response, whereas an inactive analog of genistein (diadzein) or inhibitors of serine/threonine kinases have no effect (5). The tyrosine kinase activity appears to lie downstream from the empty Ca²⁺ pool because tyrosine kinase inhibitors also block the Ca²⁺ entry stimulated by thapsigargin (5), an agent that directly empties the Ca²⁺ pools by inhibiting the Ca²⁺-ATPase that pumps Ca²⁺ into the internal stores.

To investigate the identity of the tyrosine kinase involved in regulating SOCs, we first had to identify the tyrosine kinases activated in response to BK stimulation. We recently reported (6) that in fibroblasts BK stimulates the tyrosine kinase activity of pp60^c-src (c-src). With this information in hand, we turned to the investigation of whether this tyrosine kinase is involved in the regulation of SOCs. To test for the involvement of c-src in the regulation of SOCs, we chose to utilize fibroblast lines that do not express the c-src protein tyrosine kinase as a result of gene disruption by homologous recombination (7). To investigate the regulation of SOCs by c-src, the intracellular Ca²⁺ stores of either wt cells or Src cells were emptied by various methods in a nominally Ca²⁺-free buffer, and the influx of Ca²⁺ was monitored following addition of Ca²⁺ to the external medium. The absence of c-src produced a significant decrease in the Ca²⁺ influx in response to both bradykinin and thapsigargin. This could be reversed by expression of c-src in Src cells.

EXPERIMENTAL PROCEDURES

The wt and src/src (Src) cell lines were derived by the spontaneous immortalization of mouse embryo fibroblasts prepared from either wild type mice or mice homozygous for a disruption in the c-src gene. These nonclonal cell populations were kindly provided by Philippe Soriano (Fred Hutchinson Cancer Center, Seattle, WA). The chicken c-src plasmid was kindly provided by David Shalloway (8). The plasmid carrying hygromycin resistance was obtained from Invitrogen (San Diego, CA). Avian-specific monoclonal anti-c-src antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal anti-c-src antibodies (mAb 327) were obtained from Oncogene Sciences (Uniondale, NY). Horseradish peroxidase-labeled secondary antibodies were purchased from Promega (Madison, WI). Protein assay kits and ECL reagents were obtained from Pierce.

Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin in a 5% CO₂ incubator. For measurements of intracellular calcium concentration, cells were plated onto coverslips 2-5 days prior to experiments. For Western blotting, the cells were plated onto 100-mm dishes.

Cell lysates were prepared by treating cells with 0.5% SDS/8 M urea. The lysates were freeze-thawed three times to reduce viscosity. The protein concentration was determined by the BCA method (Pierce). The protein samples were mixed with equal volume of 2 × SDS sample buffer (1 × = 62.5 mM Tris, pH 6.8, 1% SDS, 0.001% pyronin-Y, 10% glycerol, 5 mM 2-mercaptoethanol), boiled for 3 min, and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes, and nonspecific binding sites were blocked by incubating the membranes in TBS (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM KCl) containing 3% bovine serum albumin and 0.2% Nonidet P-40 for 1 h at room temperature. Antibodies at a concentration of 1 µg/ml were added, and the blot was incubated for 16 h at 4 °C. The membrane was washed with TBS/0.2% Nonidet P-40 and incubated for 40 min with horseradish peroxidase-labeled secondary antibody. The blot was washed five times, and the immunostaining was detected by enhanced chemiluminescence.

Cells were loaded with 5 µM fura-2 acetoxymethyl ester in HEPES-buffered Hanks' balanced salt solution (HHBSS) + 1 mg/ml bovine serum albumin + 0.025% Pluronic F127 detergent for 30 min at room temperature and incubated without fura-2 acetoxymethyl ester in HHBSS for 30 min, and the intracellular [Ca²⁺] was monitored as described previously (1). All traces represent the average response of 300-400 cells on a nearly confluent coverslip. Although data shown here are from nearly confluent coverslips, measurements on lower density coverslips showed that there was no density dependence of the results. Cells perfused in "nominally" Ca²⁺-free HHBSS prepared as described previously (1) are still tightly adherent and can be vigorously perfused for extended time periods.

Establishment of Src Fibroblasts Stably Expressing Chicken c-src

Fibroblasts deficient in c-src were cotransfected with chicken c-src and a plasmid carrying hygromycin resistance (pCEN4). After 2 days, cells were cultured in the presence of 250 µg/ml hygromycin for 24 h. The selection pressure was removed, and cells were cultured in normal medium. A week later cells were reselected with 150 µg/ml hygromycin for 2 days. Approximately 200 clones survived. All of the surviving clones were harvested, mixed together, and expanded to generate a heterogenous population of cells expressing chicken c-src. Cells were periodically put under selection pressure.

RESULTS AND DISCUSSION

BK-stimulated Ca²⁺ Entry in wt Fibroblasts and Src Fibroblasts

For the initial studies, cells were challenged with 100 nM BK in a nominally Ca²⁺-free external medium. The Ca²⁺ peak under these conditions (Fig. 1) is due to the emptying of inositol trisphosphate-sensitive intracellular stores (1). The difference in the height of initial peaks in individual experiments generally is due to the fact that some fibroblasts in the field had delayed response to BK, thereby producing a wider, lower peak Ca²⁺ response. Analysis of multiple coverslips (n = 12) indicates that the areas under the initial BK-stimulated peaks were not significantly different in Src and wt fibroblasts, indicating that the amounts of Ca²⁺ mobilized in the two cell lines were approximately equal. This suggests that the loss of c-src does not disrupt the signaling between the BK receptor and the Ca²⁺ stores.

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When the release of internal Ca²⁺ stores was complete and the [Ca²⁺]_i returned to base line, Ca²⁺ was added externally in the continuous presence of BK. Under these conditions the observed rise in [Ca²⁺]_i is due to capacitative Ca²⁺ influx; the initial slope of this Ca²⁺ rise was used as a measure of Ca²⁺ influx. As observed in Fig. 1, a dramatic Ca²⁺ influx was observed in BK-stimulated wild type cells. In cells that were incubated with Ca²⁺-free medium without BK, the influx of Ca²⁺ initiated by Ca²⁺ addition was only 5% of that observed in the presence of BK (data not shown). In contrast to results in wt cells, BK induced a significantly lower capacitative Ca²⁺ entry in fibroblasts deficient in c-src, as judged by the initial slope of the Ca²⁺ trace (28 ± 10% of the slope observed in wt cells, n = 12; this value was significantly different from the wt value based on a t test, p < 0.0001). Similar experiments utilizing Ba²⁺ to monitor capacitative Ca²⁺ entry also showed dramatic differences between wt and Src cells (data not shown). Because Ba²⁺ is not pumped by Ca²⁺-ATPases, the reduced Ca²⁺ entry observed in Src cells is not due to an increased Ca²⁺ pump activity but is the result of decreased capacitative Ca²⁺ influx. This observation suggests that c-src may be involved in the regulation of Ca²⁺ entry in response to BK.

Although we have demonstrated that BK-induced Ca²⁺ entry is significantly suppressed in Src fibroblasts, there does appear to be capacitative Ca²⁺ entry in Src cells. This residual Ca²⁺ influx may be due to either regulation by other tyrosine kinases or to regulation by a tyrosine kinase-independent mechanism. It has been shown that other c-src tyrosine kinase family members (e.g. c-fyn and c-yes) have some overlapping activities with c-src, which suggests the possibility that c-fyn or c-yes could also participate in the regulation of SOCs. This possibility is supported by experiments using genistein, a tyrosine kinase inhibitor that further suppressed the residual capacitative Ca²⁺ entry in Src cells (data not shown).

Thapsigargin-stimulated Ca²⁺ Entry in wt Fibroblasts and Src Fibroblasts

To exclude the possibility that c-src might regulate events in the BK signaling pathway that lie upstream of the intracellular Ca²⁺ stores, we have also examined capacitative Ca²⁺ influx in Src cells stimulated with thapsigargin (TG), a potent Ca²⁺-ATPase inhibitor (Fig. 2). Following TG-induced Ca²⁺ store depletion, Ca²⁺ influx was initiated by the addition of Ca²⁺ to the external media. Analysis of multiple wt and Src coverslips (n = 7) indicates that the total amount of Ca²⁺ mobilized by thapsigargin is not statistically different in Src and wt fibroblasts. As was the case for BK, thapsigargin-induced Ca²⁺ influx is significantly lower in fibroblasts deficient in c-src than that measured in wt fibroblasts (30 ± 15% of the slope observed in wt cells, n = 7; value significantly different from wt value based on a t test, p < 0.005), suggesting that the effect of c-src in the regulation of SOCs is downstream from the depletion of intracellular Ca²⁺ stores. The fact that Ca²⁺ entry is lower in Src fibroblasts in response to both BK and TG could mean either that c-src is involved in regulation of Ca²⁺ entry following store depletion or that the level of Ca²⁺ entry was coincidentally lower in the independently derived Src cell lines. To distinguish between these possibilities, we established Src fibroblasts stably expressing chicken c-src, thus eliminating the possibility that the independently derived Src and wt cells are different because they fortuitously express different levels of regulatory proteins or SOCs.

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Stable Transfection of c-src into Src Fibroblasts

To express c-src in Src fibroblasts, we chose chicken c-src, because this src protein was shown to have little or no transforming activity when overexpressed in NIH3T3 fibroblasts, compared with v-src and mouse c-src (8-11). We did not want to transform the cells, because transformation very often results in the overexpression or hyperphosphorylation of proteins involved in important signaling processes. The expression of c-src in Src fibroblasts posed some technical difficulties because these cells are difficult to transfect transiently. Therefore, we generated a population of cells that stably expresses the protein (see "Experimental Procedures"). The expression of chicken c-src was detected by immunostaining and also by Western blotting using the avian specific anti-c-src antibodies (Fig. 3). Western blots performed with mAb 327 demonstrated a 3-4-fold higher level of staining in the c-src transfected cells compared with wt cells (data not shown), although without knowing the relative specificity of mAb 327 for chicken versus mouse c-src, it is not clear whether this represents a 3-4-fold difference in protein expression.

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BK- and Thapsigargin-stimulated Ca²⁺ Entry in Src and Fibroblasts Stably Expressing Chicken c-src

When capacitative Ca²⁺ entry was monitored following Ca²⁺ pool depletion with BK, it was observed that stable expression of chicken c-src significantly enhanced the capacitative Ca²⁺ influx (Fig. 4). The expression of chicken c-src resulted in a 2.8 ± 0.2-fold increase (n = 7) in the initial slopes of the Ca²⁺ traces at the addition of external Ca²⁺ (value is significantly higher than Src value based on a t test, p < 0.001). In addition, when intracellular Ca²⁺ stores were depleted by treating cells with thapsigargin (1 µM), the capacitative Ca²⁺ influx was greatly enhanced in the cells expressing chicken c-src (2.7 ± 0.6-fold increase, n = 9; value is significantly higher than Src value based on a t test, p < 0.01) (Fig. 5). Although the initial BK-stimulated release of Ca²⁺ was frequently higher in the cells transfected with chicken c-src than in the Src cells, this does not explain the higher Ca²⁺ influx in the transfected cells. First, although measurements of the initial peak in chicken c-src transfected cells varied widely in height between individual coverslips, there was no correlation between first peak height and size of Ca²⁺ influx. Second, the TG-stimulated peaks frequently had the opposite relationship (although this was not the case in Fig. 5, the first peak height was often higher in Src fibroblasts than in the cells transfected with chicken c-src), and Ca²⁺ fluxes were still higher than in Src cells. Therefore, these observations strongly support our proposal that c-src is indeed involved in the regulation of calcium entry via store-operated calcium channels.

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The BK-induced capacitative Ca²⁺ entry in c-src transfected cells was 78 ± 11% of the signal observed in the wild type cells. In the case of TG-induced Ca²⁺ entry we observed 82 ± 18% restoration. Immunostaining of hygromycin-selected cells showed that we could detect strong expression of avian c-src in 25% of the cells, intermediate expression in 30% of the cells, and little or no chicken c-src staining in 45% of the cells (data not shown). Thus, because of the selection method used, not all of the cells in this heterogenous population express chicken c-src. Cells were selected based on their expression of an antibiotic resistance gene marker; thus some percentage of cells express the antibiotic resistance gene and no c-src or low levels of c-src.

Even though these data support the hypothesis that c-src may play an important role in the regulation of Ca²⁺ entry via store-operated calcium channels, we cannot exclude the possibility that c-src might be acting via a longer term mechanism, such as by controlling the level of expression of SOCs. However, our previous data (5) indicate that tyrosine kinase inhibitors reduce capacitative Ca²⁺ influx within a matter of minutes of their addition, a finding that supports the involvement of tyrosine kinases in a short term, regulatory role. However, based on the observation that the SH2 and SH3 domains but not the kinase activity of c-src are important for the effect of c-src on the rate of cell spreading on fibronectin (12), we cannot rule out the possibility that the c-src effect we observe might be independent of the src kinase domain. In the future, we plan to express c-src constructs with mutations in the kinase, SH2, and SH3 domains in Src cells to determine in more detail the importance of these domains in the regulation of Ca²⁺ entry.

Although the exact mechanism for c-src regulation of capacitative Ca²⁺ entry is not known, it appears that the decrease in Ca²⁺ entry in Src cells is not the result of a reduction in the membrane potential and therefore a decrease in the driving force for Ca²⁺ entry. Hyperpolarization of Src and wild type cells, by the addition of valinomycin in a 6 mM K⁺ medium, did not alter the fact that wild type cells had a dramatically higher level of Ca²⁺ entry than those measured in Src cells (data not shown).

There is considerable evidence that protein phosphorylation can regulate ion channels. Ion channels are known to be regulated and directly phosphorylated by a number of serine/threonine kinases, such as protein kinase C, calmodulin-dependent kinase, and cyclic AMP-dependent protein kinase (13). Some recent studies suggest that tyrosine phosphorylation can also regulate channel activities. For example, tyrosine phosphorylation regulates the channel activities of N-methyl-D-aspartate receptor (14), a brain- and heart-specific delayed rectifier-type potassium channel (15), and a voltage-dependent n-type K⁺ channel (Kv1.3) (16). A recently cloned focal adhesion kinase family member, PYK2, directly tyrosine phosphorylates a potassium channel (Kv1.2) and inhibits currents elicited by phorbol myristyl acetate (17). In regard to the regulation of SOCs, several studies suggest that serine and threonine phosphorylation may inhibit capacitative Ca²⁺ entry (18, 19), and a number of studies utilizing tyrosine kinase inhibitors (5, 20-23) support our initial proposal (5) for a role of tyrosine kinases in the activation of SOCs. Further studies are required to determine whether the effect of c-src on the regulation of capacitative Ca²⁺ entry is a direct effect on the store-operated Ca²⁺ channel, as seen for hKv1.5 and N-methyl-D-aspartate channels, or an indirect effect via other c-src substrates.