Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+ pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+ stored in intracellular pools plays an important role in this regulation.
Raf-1; mitogen-activated protein kinase; Fyn; protein phosphatase
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INTRODUCTION |
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VARIOUS GROWTH FACTORS AND G protein-linked agonists initiate the release of Ca2+ from intracellular stores by generating the second messenger D-myo-inositol 1,4,5-trisphosphate. An influx of extracellular Ca2+ also occurs, but the mechanism has not yet been fully elucidated. Recently, the capacitive Ca2+ entry model has been proposed to explain the hormone-induced activation of Ca2+ influx (36). This model predicts that depletion of intracellular Ca2+ pools opens store-operated Ca2+ channels in the plasma membrane. This has been suggested to occur via the activation of one or more tyrosine kinases (26, 41). Conversely, Ca2+ store repletion may activate tyrosine phosphatases (42, 51). These observations imply that the degree of filling of the hormone-sensitive Ca2+ pools is involved in the regulation of protein tyrosine phosphorylation within the cell.
The extracellular signal-regulated protein kinase (ERK) cascade is an important signaling pathway involved in cell growth and development that is also controlled by the activity of tyrosine kinases and phosphatases. Receptor and nonreceptor tyrosine kinases constitute initiating events of the ERK signaling cascade (1, 32). These tyrosine kinases are involved in the phosphorylation of the adaptor protein Shc. A second adaptor protein, growth factor receptor-bound protein 2 (GRB2), binds to tyrosine phosphorylated Shc through its SH2 domain (38), bringing the guanine nucleotide exchange factor son of sevenless to the membrane, where it causes activation of Ras (27). Ras, in turn, activates Raf-1, a serine/threonine kinase (31), which phosphorylates and activates the dual-specificity protein kinase mitogen-activated protein kinase (MAP kinase) kinase (MEK) (25). MEK, by phosphorylating ERK on tyrosine and threonine residues, is the direct upstream activator of ERK (12). Although the activation of Src kinases is believed to be the major upstream pathway for Ras-dependent activation of ERK, previous studies have also demonstrated the involvement of Ca2+ in this pathway (11, 14, 37).
ERK is inactivated by protein phosphatases in the cell. There are several classes of phosphatases, including serine/threonine phosphatases, tyrosine phosphatases, and dual-specificity phosphatases, some of which have been shown to directly dephosphorylate and inactivate ERK in vitro (3, 22, 40). Furthermore, activation of ERK in intact cells occurs either when protein phosphatase 2A (PP-2A) and protein phosphatase 1 (PP-1) are inhibited with okadaic acid (4, 10) or when protein tyrosine phosphatases (PTPases) are inhibited with pervanadate (56). These findings indicate that phosphatases are involved in the suppression of constitutive ERK activity. In contrast, the dual-specificity phosphatases, such as MAP kinase phosphatase-1 (MKP-1), are the products of immediate-early genes that are induced following mitogenic stimulation and contribute to negative feedback regulation. Changes in cellular Ca2+ have been implicated in the induction of MKP-1, but factors controlling the activity of these phosphatases have not been extensively characterized.
The present study was undertaken to investigate the Ca2+ dependence of ERK activation in rat liver epithelial (WB) cells. A novel mechanism for the regulation of the ERK pathway is described in which Ca2+ pool depletion inactivates various protein phosphatases, resulting in an activation of the tyrosine kinase Fyn and an increase of ERK activity in WB cells. The potential significance of this form of Ca2+ regulation was confirmed by showing that pretreatment of WB cells with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM augmented the activation of Fyn, Raf-1, and ERK by ANG II and blocked the induction of MKP-1.
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MATERIALS AND METHODS |
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Materials.
The monoclonal antibody directed against
p42ERK and ERK substrate peptide
were purchased from Upstate Biotechnology (Lake Placid, NY).
Anti-ACTIVE-MAP kinase was from Promega (Madison, WI). Antibodies to
Raf-1 and MKP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antiserum to Fyn was a generous gift of A. Y. Tsygankov (Temple University, Philadelphia, PA). Thapsigargin and okadaic acid were purchased from LC Laboratories (Woburn, MA). Genistein and ionomycin were from Calbiochem (La Jolla, CA), and EGTA was purchased from Sigma
Chemical (St. Louis, MO). Radioisotope-labeled ATP
([-32P]ATP) was
obtained from Amersham. Fura 2-AM and BAPTA-AM were from Molecular
Probes (Eugene, OR). A plasmid encoding an inactive glutathione
S-transferase (GST)-MEK-1 was provided
by Michael J. Weber (University of Virginia, Charlottesville, VA).
Cell culture. WB cells are an epithelial cell line that was originally isolated from the liver of an adult Fisher rat (48). The cells were plated onto 100-mm tissue culture plates and incubated in Richter's improved essential medium containing L-glutamine and insulin (Irvine Scientific, Santa Ana, CA) plus 10% fetal bovine serum until confluent. Cells were made quiescent by incubation for 24 h in Richter's medium without serum before the start of the experiment. Cells were used between passages 20 and 40.
Western blotting. Serum-starved WB cells were treated with or without agonist as indicated. Cells were scraped into lysis buffer containing 10 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. Cell lysates were obtained by solubilization of the cells for 30-60 min in lysis buffer, and insoluble material was removed by centrifugation at 16,000 rpm for 5 min at 4°C. The protein concentration in the supernatant was measured by the method of Bradford (7) using BSA as a standard. Equivalent amounts of protein were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Equal protein loading and the efficiency of protein transfer were assessed by staining the nitrocellulose membranes with Ponceau S. Nitrocellulose membranes were blocked with 3% (wt/vol) BSA in PBS containing 0.1% Tween 20 (PBST) for 1 h and then incubated with an antibody directed against MKP-1, p42ERK, or phosphorylated MAP kinase. Nitrocellulose membranes were washed three times with PBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Protein bands were visualized by enhanced chemiluminescence (Amersham). For the gel shift assay, ERK activation was determined by the appearance of a slower migrating band in gel electrophoresis due to phosphorylation of specific threonine and tyrosine residues.
Immune complex ERK assay.
After stimulation, WB cells were solubilized with lysis buffer, and
precleared cell lysates containing equal amounts of protein were
incubated with polyclonal anti-ERK2 antibody for 1 h on ice, followed
by an incubation with protein A-agarose with rotation for 1 h at
4°C. Preimmune serum was used as the negative control. Immunoprecipitates were washed once with lysis buffer, twice with modified RIPA buffer [10 mM MOPS (pH 7.0), 150 mM NaCl, 0.1%
SDS, 1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, and 1 mM
Na3VO4]
and twice with kinase buffer [10 mM PIPES (pH 7.0) and 10 mM
MgCl2]. The reactions were
carried out by addition of 5 µM
[-32P]ATP (10 Ci/mmol) to 5 µg/ml myelin basic protein (MBP) in the kinase buffer
at 30°C for 15 min and stopped by heating at 95°C for 5 min
after the addition of Laemmli sample buffer. The kinase assay samples
were subjected to 15% SDS-PAGE, followed by gel drying and exposure to
X-ray film at
86°C. The results were analyzed by
densitometry, with the intensity of the autoradiograms kept in the
linear range of exposures.
Raf-1 assay.
Stimulation of WB cells and immunoprecipitation of Raf-1 were carried
out as described above using an antibody against Raf-1. An irrelevant
rabbit antibody was used as a negative control. Immunoprecipitates were
washed three times with lysis buffer and then twice with kinase buffer.
The reaction was initiated by addition of 5 µM
[-32P]ATP (10 Ci/mmol) to 10 µg/ml GST-MEK in the kinase buffer at 30°C for 10 min and stopped by heating at 95°C for 5 min after the addition of
Laemmli sample buffer. The kinase assay samples were subjected to 10%
SDS-PAGE and analyzed as described for the ERK assay.
Fyn kinase assay.
Fyn was immunoprecipitated from WB cell lysates prepared as described
above using anti-Fyn serum. The antibody was raised in rabbits against
the unique region of murine
p60fyn (amino acids 6-84)
and does not recognize other Src family kinases (9, 49).
Immunoprecipitates were washed as described for the Raf-1 assay, and
the kinase reaction was initiated by addition of 5 µM
[-32P]ATP (10 Ci/mmol) to 2 µg acid-denatured enolase in kinase buffer at 30°C
for 5 min. The assay samples were subjected to 10% SDS-PAGE and
analyzed as described above.
Phosphatase activity assay. PP-2A activity was measured in whole cell lysates of WB cells using the serine/threonine phosphatase assay system (Promega). PTPase activity was similarly measured with the tyrosine phosphatase assay system (Promega).
Briefly, WB cells were grown to confluence, starved for 20-24 h, and incubated with 3 mM EGTA for up to 30 min. Cells were washed twice in ice-cold Tris-buffered saline and lysed in 200 µl of lysis buffer containing 25 mM Tris · HCl (pH 7.5), 2 mM EDTA, 10 mMMeasurement of DNA synthesis. DNA synthesis was measured by an immunocytochemical method, which assays for the incorporation of bromodeoxyuridine (BrdU) into nuclei (50). WB cells were plated onto coverslips in six-well plates, and the cells were grown to 80% confluence before being serum starved for 24 h. The cells were incubated with okadaic acid or sodium vanadate for 24 h, and the incorporation of BrdU was measured by using a commercial kit from Calbiochem (Cambridge, MA).
Measurement of cytosolic free Ca2+. Intracellular Ca2+ was measured in single cells using either fluorescence video microscopy with digital imaging analysis (Compix) or fluorescence microscopy of single cells as described previously (5).
Statistical analysis. Statistical analysis was performed by Student's t-test using SigmaSTAT software. Data are expressed as means ± SE. Differences with a P of 0.05 were considered statistically significant.
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RESULTS |
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Ca2+ store
depletion activates ERK in WB cells.
It has been suggested that an increase in intracellular free
Ca2+ is involved in the activation
of the ERK pathway, but the mechanism has not been clarified (39).
Thapsigargin, an endoplasmic reticulum (ER)
Ca2+-ATPase inhibitor, has been
extensively used to produce an increase in cytosolic free
Ca2+ without the generation of
other second messengers. The mechanism of its action is to block
Ca2+ uptake into the ER, which
gradually becomes depleted of Ca2+
due to an unopposed Ca2+ leak
(47). To elucidate the role of
Ca2+ in ERK activation, WB cells
were treated with thapsigargin for up to 30 min, and the activation of
immunoprecipitated ERK2 was assessed by three separate assays. For the
first, ERK activity was measured by the incorporation of
32P from
[-32P]ATP into MBP
(Fig.
1A).
ERK activation is associated with phosphorylation of the enzyme on
threonine and tyrosine residues, which causes an increase in its
electrophoretic mobility. Therefore, ERK activity was also evaluated by
the gel shift assay (Fig. 1B). For
the third method, measurements were made of the amount of
phosphorylated ERK in cell lysates, as detected by an antibody directed
against the phosphorylated forms of ERK1 and ERK2 (Fig.
1C). All three methods
demonstrated that treatment of WB cells with thapsigargin caused an
increase in ERK activity. Maximum activity varied between 15 and 30 min
for the different experiments shown in Fig. 1 and was considerably
slower than the rise in intracellular free
Ca2+ produced by thapsigargin
stimulation. This was maximal at ~1 min and gradually declined to
basal Ca2+ levels by 15 min (Fig.
2A).
Occasionally, as in Fig. 2B, maximum ERK activity after thapsigargin addition occurred before 15 min. Densitometric quantitation of ERK activity measured in four experiments similar to Fig. 1A showed a three-
to fourfold increase in ERK activity after 15 or 30 min. These data
suggest that an increase in cytosolic free
Ca2+ per se may not be directly
responsible for the thapsigargin-induced increase in ERK activity.
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Genistein sensitivity of ERK activation induced by Ca2+ pool depletion. Recent studies suggest that depletion of the intracellular Ca2+ pool leads to an activation of tyrosine kinases that stimulate Ca2+ influx, although the mechanism remains unknown (26, 41). To determine whether the activation of ERK by Ca2+ store depletion involved a tyrosine kinase pathway, WB cells were pretreated with 100 µM genistein for 30 min before the addition of 3 mM EGTA. Figure 4A shows that genistein almost completely inhibited the EGTA-induced activation of ERK, indicating that intracellular Ca2+ depletion is associated with the activation of a tyrosine kinase upstream of ERK in the ERK pathway. Also, the increased activity of ERK induced by thapsigargin in the presence or absence of BAPTA was inhibited by genistein (Fig. 4B). Genistein also blocked the increased ERK activity observed in cells treated with BAPTA alone. Similar results were obtained when ERK activation was measured by the gel shift assay and by the amount of phosphorylated, activated ERK in immunoblots (data not shown). These findings support the conclusion that the activation of ERK induced by Ca2+ pool depletion takes place through a tyrosine kinase-sensitive pathway in WB cells.
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Ca2+ pool depletion increases the activity of Fyn and Raf-1. Fyn and Yes, two members of the Src family of tyrosine kinases, have previously been shown to be upstream kinases of the ERK pathway in WB cells (50). Because Ca2+ pool depletion with thapsigargin increased ERK activity in a genistein-sensitive manner, the activation of Fyn and Yes was examined in WB cells treated with 2 µM thapsigargin for various times. These Src family kinases were immunoprecipitated and subjected to an in vitro kinase assay using enolase as the substrate. Figure 5A shows that thapsigargin caused a rapid threefold activation of Fyn, and Fig. 5B shows that the thapsigargin-induced increase in Fyn activity was unaffected by buffering the intracellular free Ca2+ with BAPTA, although BAPTA alone caused a twofold activation of Fyn. In contrast, thapsigargin had no effect on the activity of Yes in WB cells (unpublished observations). These results suggest that there is a specificity of function between Fyn and Yes and that only Fyn tyrosine kinase is activated by depletion of the thapsigargin-sensitive Ca2+ pool.
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Ca2+ depletion increases PP-2A and PTPase activities in WB cells. Intracellular Ca2+ pools have been suggested to regulate the activity of protein phosphatases within the cell (42, 51). Treatment of WB cells with okadaic acid, an inhibitor of the serine/threonine phosphatases PP-2A and PP-1, and vanadate, a tyrosine phosphatase inhibitor, caused an increase in ERK activity in WB cells (data not shown). Moreover, 10 nM okadaic acid or 10 µM vanadate caused a twofold increase of DNA synthesis (Table 1). These results suggest that one or more phosphatases contribute to maintenance of a low basal level of ERK activity and cell proliferation in the absence of growth factors. The possibility that Ca2+ depletion might also influence the overall phosphatase activity in the cytosol was evaluated by measuring the activities of PP-2A and PTPase in cells treated with 3 mM EGTA (cf. Fig. 3). Ca2+ depletion resulted in a time-dependent decrease in the activity of PP-2A, as measured by phosphate loss from a phosphoserine peptide substrate (Fig. 6, bottom). Inhibition was maximal at 5 min, and phosphatase activity remained decreased for up to 30 min. The peptide substrate used was not a substrate for PP-1, and addition of okadaic acid to the reaction mixture fully blocked the phosphatase activity, indicating it was due to PP-2A (data not shown). Additionally, there was a similar time-dependent decrease in PTPase activity in WB cells treated for up to 30 min with EGTA (Fig. 6, top). These data suggest that intracellular Ca2+ depletion results in the inactivation of both PP-2A and PTPases, two groups of phosphatases that are known to dephosphorylate a variety of proteins, including the Src family kinase members MEK and ERK.
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Intracellular Ca2+ chelation augments ANG II-induced activation of the ERK pathway and represses expression of MKP-1 by ANG II. Mobilization of Ca2+ from the ER is a well-established response to numerous hormones and growth factors. Therefore, the Ca2+ chelator BAPTA was used to investigate the involvement of Ca2+ in the ERK pathway initiated by the G protein-coupled receptor agonist ANG II. Pretreatment of cells with BAPTA-AM for 15 min caused a greatly augmented activation of ERK by ANG II, which was statistically significant after 30 and 60 min (Fig. 7A), but completely blocked the rise in intracellular free Ca2+ normally obtained with ANG II (54). Pretreatment of the cells with BAPTA also augmented the ANG II-induced activation of Fyn and Raf-1, even after 30 s (Fig. 7, B and C). Similarly, Graves et al. (21) have shown that the ANG II-induced activation of ERK and p90RSK was augmented in GN4 cells pretreated with similar concentrations of BAPTA. The probable reason for this effect is that buffering the cytosolic Ca2+ with BAPTA leads to a diminished refilling of the Ca2+ pools in these cells after stimulation with ANG II, thereby causing a greater Ca2+ depletion relative to conditions with ANG II and no BAPTA.
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DISCUSSION |
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The amount of Ca2+ within the ER has been shown to affect several cellular functions, including translation, folding, processing and assembly of proteins (30), protein synthesis (15, 29), cell proliferation (20), Ca2+-independent PLA2 (iPLA2) activity (53), and Ca2+ influx (6). In the present study with WB cells, regulation of the ERK pathway was investigated after depletion of the ER Ca2+ pools by two different methods. In the first method, Ca2+ stores were depleted with thapsigargin, which produced an increase in ERK activity. This was not due to a rise in cytosolic free Ca2+, since similar findings were obtained when the thapsigargin-induced increase in cytosolic free Ca2+ was blocked by introducing the Ca2+ chelator BAPTA into the cells. In the second method, intracellular Ca2+ was depleted by the addition of excess EGTA to the medium. This caused the depletion of cellular Ca2+ pools as well as a fall in cytosolic free Ca2+. Because ERK was also activated under these conditions, it is evident that Ca2+ store emptying as opposed to an elevation of cytosolic Ca2+ was responsible for the increase in ERK activity in WB cells under these conditions (Figs. 2-4).
In most electrically nonexcitable cells, Ca2+ store depletion stimulates Ca2+ influx via the activation of store-operated Ca2+ channels (26, 41). Furthermore, under a number of conditions, Ca2+ influx has been shown to be sensitive to the tyrosine kinase inhibitor genistein (6, 44, 51). This finding has led to the hypothesis that the release of sequestered Ca2+ activates a tyrosine kinase, whereas repletion of Ca2+ stores activates one or more phosphatases (42, 51). Moreover, it has also been demonstrated that activation of store-operated Ca2+ channels and the resultant Ca2+ influx are governed by the status of Ca2+ within the ER (23). In WB cells, we observed that the Ca2+ depletion-induced activation of ERK was sensitive to inhibition by genistein (Fig. 4). Furthermore, data presented here suggest that the tyrosine kinase Fyn may be specifically involved in the activation of ERK under conditions of Ca2+ store depletion, since its activity was increased after thapsigargin treatment of WB cells and since this activity was unaffected by pretreatment of the cells with the Ca2+ chelator BAPTA (Fig. 5). This coincides with the fact that Fyn is an upstream component of the ERK pathway initiated by ANG II in WB cells (50). A decreased activity of two groups of phosphatases, PP-2A and PTPases, was also observed after treatment of the cells with EGTA (Fig. 6). Therefore, it would seem that signals transmitted by depleted Ca2+ stores to enhance ERK activity involve the activation of the protein tyrosine kinase Fyn and the inactivation of at least two types of protein phosphatases, PP-2A and PTPase. The involvement of both Fyn and these phosphatases in the ERK pathway may contribute to the delayed activation that was observed following Ca2+ pool depletion.
Although it has been suggested that intracellular Ca2+ stores are involved in the regulation of cellular phosphatases, the present study extends this concept by demonstrating a reduction in PP-2A and PTPase activity by Ca2+ store depletion. However, the mechanism by which the degree of filling of the Ca2+ stores regulates the phosphatase activities remains unclear. A possible mechanism is suggested by the fact that one of the main PTPases, PTP-1B, contains a COOH-terminal hydrophobic segment that targets it to the ER (17). Agents that increase intracellular Ca2+ promote the cleavage of PTP-1B by calpain, resulting in its translocation to the cytosol and subsequent activation (17). In turn, depletion of stored Ca2+ may also affect the activity of PTP-1B by promoting its retention in the ER.
Protein phosphatases generally have a negative effect on cell proliferation and appear to be active in resting cells, thereby providing a mechanism for the maintenance of growth arrest by limiting the activities of growth-promoting kinases (2). This is in agreement with the results showing that inhibition of phosphatase activity with okadaic acid and/or vanadate caused an increase in the rate of DNA synthesis (Table 1). Because an inactivation of PP-2A and PTPases occurs with intracellular Ca2+ depletion, one or more phosphatases that are normally involved in the maintenance of a low basal level of ERK activity in quiescent cells probably become inhibited. Pervanadate has been shown to cause an activation of ERK as well as of the upstream kinases MEK and Raf-1 (56). Furthermore, pervanadate induced tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with GRB2. This indicates that pervanadate-sensitive phosphatases are interacting with upstream components of the ERK pathway, possibly also causing an activation of Src family kinases, whereas okadaic acid-sensitive phosphatases may directly dephosphorylate MEK and/or ERK (56).
Control of tyrosine kinases and phosphatases by the amount of stored Ca2+ may also be critical for regulating ERK activity following hormonal stimulation. Previously, the involvement of Ca2+ in the ERK pathway initiated by various agonists in different cells has been shown to be dependent on the cell characteristics and the agonist used (16, 18). For example, in cardiac myocytes, ANG II-induced activation of ERK appeared to be dependent on a rise in cytosolic Ca2+ (39), whereas carbachol-induced activation of ERK was minimally affected by BAPTA or EGTA pretreatment in Rat 1a fibroblasts (34). The present study suggests that intracellular Ca2+ pool content may play a novel role in the hormonal regulation of the ERK pathway. Under resting conditions, ERK activity appears to be restrained by phosphatases that are constitutively active in the quiescent cell. When stimulated with ANG II, intracellular Ca2+ is mobilized, resulting in Ca2+ store depletion. This normally is a transient phenomenon, since the release of stored Ca2+ initiates Ca2+ influx, which leads to a partial refilling of the Ca2+ pools (26, 41). It is proposed that the initial reduction of Ca2+ in the ER pool activates Fyn and inactivates PP-2A and PTPases, both of which may contribute to the activation of ERK observed after ANG II stimulation. Subsequently, the Ca2+ pools are refilled and PP-2A and PTPases are reactivated. Concurrently, the initial transient rise in intracellular free Ca2+ induces the expression of MKP-1. Together, these events affect the duration of activated ERK, as a new steady state is reached between the activities of the various protein kinases and phosphatases affecting different steps in the ERK pathway.
ERK is directly involved in the initiation of mitogenesis as a
consequence of the phosphorylation and activation of a variety of
transcription factors. However, although the depletion of stored Ca2+ activates ERK, this alone is
insufficient for the induction of mitogenesis (20). In cells in which
there is a prolonged elevation of cytosolic
Ca2+, thapsigargin induces
apoptosis (19). In contrast, prolonged intracellular
Ca2+ pool depletion induced by
thapsigargin caused an arrest of
DDT1 MF-2 smooth muscle cells in a
G0-like state, which could only be
reversed by the synthesis of new functional
Ca2+ pump protein and subsequent
replenishment of the Ca2+ pools
(52). In our hands, thapsigargin completely blocked EGF-induced BrdU
incorporation in WB cells (data not shown), indicating that functioning
(replete) Ca2+ pools are an
absolute requirement for cells to proceed through the cell cycle.
Depletion of Ca2+ stores has been
implicated in the regulation of gene transcription and translation (8,
35). Ca2+ store depletion has been
shown to activate double-stranded RNA-dependent/regulated protein
kinase (PKR), which in turn phosphorylates and activates eukaryotic
initiation factor 2, resulting in the arrest of translation at
initiation (35, 45). Conversely, PKR has also been shown to activate
the transcription factor nuclear factor-
B (24). Additionally,
depletion of Ca2+ pools increases
the expression of genes that encode select resident ER proteins, such
as glucose-regulated protein 78 and calreticulin (28, 29). This
indicates that Ca2+ pools are
important for both the inhibition and stimulation of protein synthesis.
Therefore, the amount of Ca2+
within the ER may help to maintain a precise control over ERK activity
and consequently over protein synthesis and mitogenesis under different
conditions of either cellular stress or proliferation.
In summary, it has been established that depletion of Ca2+ stores, even in the absence of an elevation of cytosolic free Ca2+, is sufficient to induce the activation of ERK in WB cells. This apparently occurs via the activation of Fyn, and possibly via the inactivation of both serine/threonine and/or tyrosine phosphatases. However, the mechanism whereby such diverse systems as Ca2+ channels, protein kinases, phosphatases, and phospholipases such as iPLA2 respond to the Ca2+ content of intracellular Ca2+ stores remains elusive.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-15120 and DK-48494 (to J. R. Williamson), an American Diabetes Association Career Development Award (to L. Yang), and NIDDK Postdoctoral Fellowship DK-09404 (to J. A. Maloney).
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: J. R. Williamson, University of Pennsylvania, Dept. of Biochemistry and Biophysics, 601 Goddard Labs, 37th and Hamilton Walk, Philadelphia, PA 19104.
Received 27 July 1998; accepted in final form 12 October 1998.
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