(Received for publication, August 8, 1994; and in revised form, October 21, 1994)
From the
Depletion of endoplasmic reticulum (ER) Ca store by thapsigargin (Tg) in mammalian cells induces a set of ER
protein genes known as the glucose-regulated proteins. Recently, IRE1p,
a transmembrane protein postulated to have a serine/threonine kinase
activity, has been identified as required for the induction of ER
resident proteins genes in Saccharomyces cerevisiae. To
investigate whether IRE1p can stimulate mammalian grp transcription, a stable Chinese hamster ovary cell line containing
amplified copies of IRE1p has been created. The IRE1p expressing
transfectants exhibited a modest (2-fold) enhancement of both the basal
and Tg induced level of grp78 and grp94, two
coordinately regulated grp genes. Using okadaic acid as a
specific inhibitor for the endogenous serine/threonine protein
phosphatase activities, a mild (2-fold) stimulative effect was observed
for Tg induction of grp78 transcription. The okadaic acid
potentiating effect requires a 50-base pair region in the vicinity of
the grp78 TATA element. In contrast, the transcriptional
activation of grp78 by Tg is almost totally eliminated by
genistein, a tyrosine kinase inhibitor. The grp core, the C3
and C1 elements which are major Tg response elements of the rat grp78 promoter, are also major targets of the inhibitive
effects of genistein.
Thapsigargin (Tg) ()is a tumor promoter whose
intracellular target has been identified as the endoplasmic reticulum
(ER) Ca
-ATPase (Jackson et al., 1988;
Thastrup et al., 1990). Through its inhibition of the enzyme,
Tg specifically discharges the ER calcium store. Within the ER resides
a set of proteins known as the glucose-regulated proteins (GRPs) (Lee,
1992). GRP78, a 78-kDa protein also referred to as the immunoglobulin
heavy chain protein (BiP), is one of the best characterized GRPs with
protein chaperoning and calcium binding properties (Pelham, 1986;
Little et al., 1994). Although GRP78 is constitutively
expressed at a low basal level in many different tissues, its
transcription is highly inducible by a variety of stress conditions,
particularly those which perturb ER function (Lee, 1987; Watowich et al., 1988; Kozutsumi et al., 1988; Lee, 1992; Liu et al., 1992; Price et al., 1992). In mammalian
cells, one of the most potent inducers for grp78 is chronic
depletion of ER calcium stores by Tg or the calcium ionophore A23187
(Resendez et al., 1986; Drummond et al., 1987; Li et al., 1993). The induction process is independent of a rise
in cytoplasmic Ca
; furthermore, it requires a
prolonged period of (2 h) of treatment and is sensitive to
cycloheximide. Thus, the signaling mechanism for the activation of grp78 transcription involves more components than merely the
efflux of Ca
from the ER.
In examining the regulatory elements of the rat grp78 promoter which mediate the Tg and A23187 induction response, two functional domains have been identified (Wooden et al., 1991; Li et al., 1993). One region spans the promoter sequence from -159 to -110 and contains the grp core element which is conserved from yeast to human (Resendez et al., 1988) and a C3 element (Li et al., 1993). The other region spans -109 to -74 and contains a CCAAT motif (C1) most proximal to the TATA element (Wooden et al., 1991). Both regions are able to confer Tg and A23187 inducibility to a heterologous promoter upon transient transfection into mammalian cells (Li et al., 1993). Using ligation-mediated PCR, we found that specific changes in factor occupancy occurred after stress induction and that the major changes occur within the grp core element (Li et al., 1994). The factor which exhibits specific binding to this region appears to be constitutive, suggesting that during stress, the factor either undergoes a conformational change or has dissociated from inhibiting elements, resulting in the changes in the in vivo footprinting pattern.
The discovery that chronic depletion of Ca from the ER store could lead to grp78 transactivation
correlated with factor interaction changes at the grp78 promoter, raises the important question of intracellular
compartment signaling. What are the mediators of this response? The
transcription of grp78 in fibroblast cells is partially
inhibited by W-7 which inhibits
Ca
/calmodulin-regulated enzyme activities (Resendez et al., 1986). Other studies suggest an involvement of
tyrosine and serine/threonine kinases and phosphatases. In the
mammalian system, pretreatment of NIH3T3 cells with genistein, an
isoflavone which specifically inhibits a number of tyrosine kinases,
reduces Tg induction of grp78 mRNA levels, whereas okadaic
acid (OA), a specific inhibitor of serine/threonine protein
phosphatases type 1 (PP-1) and type 2A (PP-2A)
(Schönthal, 1992), enhances Tg induction of grp78 (Price et al., 1992). In the yeast system, a IRE1/Ern1 gene encoding for a transmembrane serine/threonine
kinase has recently been identified as required for the transcriptional
induction of KAR2, the yeast homologue of grp78, in
response to the accumulation of malfolded protein in the ER (Cox et
al., 1993; Mori et al., 1993). It has been postulated
that IRE1p is the proximal sensor of events in the ER and that binding
of ligands causes transduction of information across the ER membrane,
leading to activation of a specific set of transcription factors. IREp1
is of very low abundance in yeast cells. Its ligand and the involvement
of IRE1p/Ern1p in Ca
signaling of grp78 have
not yet been determined.
Since IRE1p is the first and the only serine/threonine kinase identified so far which can affect KAR2/grp78 transcription, we created a mammalian expression vector for the yeast IRE1p and selected for stable transfectants containing amplified copies of the yeast gene. We report here that expression of IRE1p in hamster ovary cells is correlated with a 2-fold increase in the basal and Tg-induced levels of the endogenous grp78 mRNA. In mammalian cells, grp78 is coordinately regulated at the transcriptional level with grp94, which encodes a 94-kDa GRP, through common cis-regulatory elements and trans-acting factors (Chang et al., 1987; Liu and Lee, 1991). As in the yeast system where IRE1 also induces other ER resident protein genes (Cox et al., 1993), the mammalian IRE1p transfectants also exhibit 2-fold increase in the basal and Tg-induced levels of grp94 mRNA.
Because of the modest effect observed with overexpression of the yeast IRE1p protein in mammalian cells and the inherent difficulties associated with expression of a heterologous protein, we investigated the respective contributions of serine/threonine protein phosphatases and tyrosine kinases toward the Tg induction of grp78 in two different mammalian cell lines. Our results indicate that genistein eliminates the response almost completely. We further demonstrate that the inhibitive effect of genistein is at the level of transcription. We found that the grp core, the C3 and C1 elements which are the major Tg response elements of the rat grp78 promoter (Li et al., 1993), are also major targets of the inhibitive effects of genistein. The tyrosine kinase activity essential for the Tg induction of grp78 is likely to be distinct from EGFR, since lavendustin-A (L-A), a potent inhibitor for EGF-associated tyrosine kinase activity (Onoda et al., 1989), is without effect.
Using OA as a specific inhibitor for serine/threonine protein phosphatase activities, a mild (about 2-fold) stimulative effect was observed for Tg induction of grp78 mRNA levels. Nonetheless, the OA stimulative effect is not mediated through the Tg response elements; rather, it requires sequences in the vicinity of the grp78 TATA element unrelated to the known OA response elements. Thus, in the mammalian system, although a serine/threonine kinase activity may potentiate slightly the grp78 Tg response, a genistein-sensitive activity acting through the Tg response elements is the critical component of the signal transduction system that leads to transcription activation of grp78.
For this purpose, the yeast IRE1p coding sequence was obtained by engineering PCR primers directly flanking its ATG translation initiation codon and the termination codon (Fig. 1). After PCR amplification using the yeast genomic clone pCS110 as a template, the 3.3-kb fragment containing the IRE1p coding sequence was subcloned into the multiple cloning site located between the Rous sarcoma virus (RSV) long terminal repeat and an SV40 poly(A) site of the DHFR expression vector pRSV-1 (Li et al., 1992). This mammalian expression vector for IRE1p is referred to as pRSV-IRE. For purpose of in vitro transcription, the same IRE1p coding sequence was subcloned into a T3 vector and is referred to as pBS-IRE.
Figure 1: Construction of the expression plasmids. Plasmid pCS110 containing the yeast IRE1 gene was used to PCR-amplify a 3.3-kb full-length of IRE1 gene with SalI and BamHI sites at two ends. The IRE1 gene was then subcloned into pRSV-1 immediately after the LTR promoter to generate the pRSV-IRE plasmid, in which there is a selection marker, DHFR gene under the control of SV40 promoter. This plasmid was used to generate the stable cell line for the expression of IRE1 gene. The IRE1 gene derived from pRSV-IRE was further subcloned into pBS to generate the pBS-IRE plasmid. The pBS-IRE was used as a template in in vitro transcription under the direction of a T3 promoter.
Prior to transfection into
mammalian cells, we established that the IRE1p coding sequence obtained
from PCR was capable of translating into a protein close to the 127-kDa
size predicted for IRE1p. In vitro transcribed IRE1p RNA was
translated in a rabbit reticulocyte system in the presence of
[S]methionine. As shown in Fig. 2A, only the sample containing the IRE1p mRNA was
capable of synthesizing a protein band of about 130 kDa. Thus, the PCR
DNA sequence is devoid of cryptic termination codons. Next, pRSV-IRE
and as a control, the vector pRSV-1, were transfected into DG44 cells,
which is a CHO cell line devoid of DHFR activity. Stable transfectants
were selected, and the transfected plasmids were amplified by
subjecting the transfected cells to increasing concentrations of MTX.
Southern blot analysis of genomic DNA prepared from these cells
revealed that at MTX concentrations of 0.2 µM and higher,
there was notable amplification of IRE1p coding sequence in the
pRSV-IRE transfectants (Fig. 2B). By comparing the band
intensities with the control plasmid ran in parallel in the same DNA
blot, we estimated the copy number of the IRE1p coding sequence in the
stable transfectants was about 50. To confirm that pRSV-IRE stable
transfectants were expressing IRE1p, total protein extract was prepared
from stable transfectants selected by 5 µM MTX, as well as
from pRSV-1 transfectants subjected to the same MTX selection. To
detect IRE1p, a Western blot was performed using antisera raised
against the N terminus of IRE1p. In the protein sample from the
pRSV-IRE transfectants, an immunoreactive band of 130 kDa was detected.
This band was not observed in the control sample from pRSV-1
transfectants (Fig. 2C). These results establish that
in the pRSV-IRE stable cell line where the IRE1p coding sequence was
highly amplified, sufficient amounts of IRE1p was produced to allow
detection by immunoreactivity with antibody specific for IREp.
Figure 2:
IRE1p expression in vitro and in vivo. A,
[S]methionine-labeled luciferase (lane
1) and IRE1p (lane 2) were synthesized in the in
vitro rabbit reticulocyte system and analyzed by 8% SDS-PAGE and
autoradiography. As a negative control (lane 3), a translation
reaction mixture was performed without any mRNA. The arrow indicates the location of
[
S]methionine-labeled IRE1p. B, pRSV-1
and pRSV-IRE were stably transfected into DG44 cell lines. Genomic DNA
were isolated from DG44 (lane 1), pRSV-1 stable transfectants
selected by 5 µM MTX (lane 2) and pRSV-IRE
transfectants selected by increasing concentrations (0.005-5.0
µM) of MTX (lanes 3-6). The DNA was
digested with EcoRI and separated on the agarose gel, blotted
onto the filter, and hybridized with the IRE1 gene. In lane 7, the pRSV-IRE plasmid was digested with EcoRI. C, the IRE1p expression in pRSV-1 (lane 1) and pRSV-IRE (lane 2) stably transfected cells selected by 5 µM MTX was analyzed by Western blot using antisera against yeast
IRE1p. The arrow indicates the location of
IREp.
Figure 3: Relative mRNA levels of grp78 and grp94 in the stably transfected cells after thapsigargin treatment. A, total RNA were isolated from pRSV-1 and pRSV-IRE stably transfected cells selected by 5 µM MTX. At 16 h prior to RNA extraction, cells were untreated(-) or treated (+) with 300 nM of thapsigargin. The RNA blots were hybridized with grp78, grp94, and 3A10 cDNA probes. The autoradiogram is shown. B, levels of grp78 and grp94 mRNA from stably transfected cells were quantitated by densitometry and normalized against the invariant 3A10 mRNA level. The mRNA levels for grp78 and grp94 from pRSV-1 stably transfected cells without treatment of thapsigargin were set as 1. The fold induction by Tg is indicated. RNA levels from cells treated with (+) or without(-) thapsigargin are plotted.
Figure 4: Effect of genistein and okadaic acid on the thapsigargin induced accumulation of grp78 mRNA. A, total cytoplasmic RNA was isolated from untreated K12 cells (C); cells treated with 300 nM thapsigargin (Tg) and cells pretreated with genistein then Tg added (G + Tg), with 100 nM of okadaic acid (OA), or pretreated with OA then Tg added (OA + Tg). RNA blot analysis for grp78 and 3A10 mRNA levels were performed. The autoradiograms are shown. B, the levels of grp78 mRNA from K12 and NIH3T3 cells after treatment with okadaic acid (OA), genistein (G), or thapsigargin (Tg) or in combinations (OA +Tg or G + Tg) were determined by densitometry and normalized against mRNA levels of 3A10. The levels of grp78 mRNA in untreated NIH3T3 or K12 cells (C) were set as 1. The fold induction for each of the treatment conditions is shown.
Figure 5: Effect of genistein and okadaic acid on the Tg induction of grp78 promoter activity. A, NIH3T3 cells were transiently transfected with(-456)CAT and subjected to treatments with OA, genistein, and Tg alone or in combinations (OA + Tg or G + Tg). The autoradiogram of the CAT assays is shown. The positions of the chloramphenicol (CM) and its acetylated forms (3Ac and 1Ac) are indicated. B, the CAT conversion in each sample was quantitated by an Ambis Radioanalytic Imaging System. The level of the promoter activity in untreated cells (C) was set as 1. The fold induction for the various treatments are shown.
Figure 6: Schematic drawings of the grp78/CAT and grp78/MCAT fusion genes used in the transient transfection assays. The 5` and 3` end points of the grp78 promoter fragments used are indicated. The positions of the core, C3, C1, and TATA elements are indicated. The small triangles indicate upstream enhancer elements. The hatched lines in the MCAT constructs represent the promoter sequence of the mouse mammary tumor virus (MMTV). The MCAT plasmid contains the promoter fragment (-105 to +104) of the MMTV fused to the CAT reporter gene. The target regions for the inhibitive effect(-) of genistein (G) and okadaic acid (OA) and stimulative effect (+) of OA during Tg induction are bracketed.
Figure 7: Differential effect of genistein and okadaic acid on the Tg induction of grp78/CAT and grp78/MCAT fusion genes. Left panels, comparison between(-355)CAT and (-375/-88)MCAT. Right panel, comparison between (-109/-MCAT and the minimal MMTV promoter driven CAT (MCAT). NIH3T3 cells were transiently transfected with these plasmids and subjected to the various treatment conditions (C, Tg, OA, G, OA + Tg, G + Tg). The autoradiograms of the CAT assays are shown. The position of chloramphenicol (CM) and its acetylated forms (3Ac and 1Ac) are shown.
We next tested (-375/-88)MCAT, which contains 287 bp of the grp78 promoter fused to a minimal MMTV promoter. The results showed that the 4-fold induction of CAT activity by Tg was completely eliminated when the cells were treated with genistein in combination with Tg, whereas genistein alone showed no effect (Table 1). Thus, genistein response elements are located within this 287-bp region. One of the target domains spans -159 to -110, since duplicate copies of this subfragment when linked to the minimal MMTV promoter (-159/-110)CAT was able to confer a 8-fold induction by Tg. However, this induction was reduced to 1.6-fold when genistein was added (Table 1). This region contains the 3`-half of core and C3 element. Another target region spans -109 to -74 and it contains the C1 element. Duplicate copies of this subfragment linked to the minimal MMTV promoter (-109/-74)CAT were able to confer a 13-fold induction by Tg. Addition of genistein and Tg completely eliminated the induction ( Fig. 7and Table 1).
To demonstrate that the inhibitive effect of genistein on grp78 transcription is not due to a general toxic effect of tyrosine kinase inhibitors and to delimit the tyrosine kinase activity targeted by genistein, we tested the effect of L-A, a potent inhibitor for EGF receptor-associated tyrosine kinase activities (Onoda et al., 1989) on(-456)CAT and(-355)CAT. Two concentrations (50 nM and 1 µM) of L-A were used. The results, as shown in Table 1, demonstrated that L-A, either added alone or in combination with Tg, did not have any effect.
Since OA exerts a potentiating effect
on(-456)CAT but not on (-375/-88)MCAT (Fig. 7), the
target for the stimulation either lies in the upstream region spanning
-456 and -375 or in the downstream region spanning
-88 to -38 (Fig. 3). The upstream region contains
basal enhancer elements and the downstream region contains the sequence
surrounding the grp78 TATA element at -55 but excludes
transcription initiation located at (+1) (Li et al.,
1993). To differentiate between these two possibilities, we first
compared the response between(-457)CAT and(-355)CAT. Our
results show that these two plasmids responded nearly identically to
the treatment of Tg and OA (Table 1). Thus, the upstream sequence
between -456 and -355 is not needed for the potentiating
effect as both plasmids showed that OA enhanced the Tg induction from
about 6-12-fold. Next the response between(-355)CAT and
(-375/-88)MCAT was compared. While both plasmids showed a
4-5-fold induction by Tg, OA in combination with Tg stimulated
the(-355)CAT activity to 11-fold and reduced
(-375/-88)MCAT activity to 2-fold ( Fig. 7and Table 1). The minimal MMTV promoter (MCAT) was relatively
unaffected by Tg or the various inhibitors, either added alone or in
combinations with Tg ( Fig. 7and Table 1). Our results
show that the OA stimulative effect of grp78 Tg induction
requires a 50-bp downstream region surrounding the grp78 TATA
sequence. Previously, we demonstrated that this same region, when fused
to a CAT reporter gene, is almost devoid of any basal promoter activity
and is not inducible by Ca depletion stress (Resendez et al., 1988).
With the recent discovery that in the S. cerevisiae a serine/threonine kinase (IRE1p) plays a critical role in
activating the ER protein gene transcription in response to malfolded
protein accumulation stress, the question arises as to whether protein
phosphorylation is also utilized as a mechanism for the induction of
the mammalian ER protein genes. In this report we focused on grp78 gene expression which is induced to high levels by thapsigargin
following chronic depletion of the ER Ca store and
provide several lines of evidence that protein phosphorylation and
dephosphorylation are involved in grp transactivation.
Our first approach is to engineer the coding sequence of IRE1p into a mammalian expression vector and create a stable CHO cell line with amplified copies. This was achieved, and a cell line harboring about 50 copies of the yeast gene was established. In these cells, sufficient amounts of IRE1p were expressed to allow identification with specific antibody against the protein. Nonetheless, under these favorable conditions, the enhancement for grp78 mRNA level in stressed and nonstressed cells was about 2-fold. Could this slight increase in grp78 mRNA levels be due to the serine/threonine kinase activity of the transfected gene? The fact that the 2-fold induction we observed is similar to that reported for S. cerevisiae when the same protein is overproduced over the endogenous level (Mori et al., 1993) suggests that this might be the physiological limit for this protein in a normal cell background. Furthermore, the co-induction of other ER protein genes such as grp94 in the IRE1p expressing cells is consistent with the action of IRE1p in yeast cells. On the other hand, expression of a heterologous protein has its inherent problems which are difficult to resolve in a mammalian system. For example, the modest enhancement of the grp mRNA levels could have been caused by improper insertion of IRE1p into the designated cellular compartment, thus diminishing the capacity of IRE1p to fully induce the grps. Another complication is that the portion of IRE1p located in the endoplasmic reticulum might not fold properly. This effect could have elicited a mild grp stress response. Although amino acids essential for the kinase activity have been identified within IRE1p (Mori et al., 1993), to create an equivalent CHO cell line with the amplified mutant genes at the same level of expression as the wild type gene to differentiate between a 2-fold effect is technically difficult, if not impossible. The functional significance of IRE1p in animal cells is best evaluated when the fold induction of the transfected wild-type protein can be manipulated so that it is sufficiently high to allow mutational analysis. This may be possible using recipient cells with the homologous mammalian gene deleted. However, this requires the confirmation that such a gene exists in mammalian cells and its subsequent isolation. Based on the current limitations, we focused our effort into examining the contribution of endogenous protein kinases and phosphatases in the Tg induction of grp78.
Potent
protein kinase inhibitors have been used widely as biochemical tools to
study the function of kinase and phosphatase activities in regulating
gene expression in mammalian cells. In this study we use a panel of
specific inhibitors to determine their effect on grp78 induction by Tg. The majority of intracellular phosphatase
activity has been attributed to PP-1 and PP-2A, and OA has the ability
to inhibit both enzyme activities. Generally, PP-2A is inhibited more
efficiently than PP-1 (Cohn, 1989). In our experiments, we discovered
that OA only exhibits a mild potentiating effect on grp78 transcription. Since the effect is more pronounced at 100 nM of OA than 50 nM(), PP-1, rather than PP-2A,
may be the affected phosphatase. OA has been reported to stimulate a
large number of gene systems, including that of the heat shock gene
(Schönthal et al., 1991; Guy et
al., 1992; Chang et al., 1993). Since OA strongly
increases the heat shock hsp70 promoter activity, it was proposed that
the heat-induced transcriptional activation of the heat shock gene is
associated with the phosphorylation of component(s) of the
transcriptional complex and that OA enhances this phosphorylation
(Chang et al., 1993). Based on the optimal concentrations of
OA, PP-2A, as opposed to PP-1, was implicated in this induction
process. Thus, although grp and hsp genes are both
stress-inducible genes and share some common induction conditions (Lee,
1987; Watowich and Morimoto, 1988), their response to OA during stress
is not identical.
In examining the grp78 promoter sequence which is required for the OA stimulative effect on Tg induction in NIH3T3 cells, we discovered that it does not act through the Tg response elements. Rather, it exerts a mild inhibitive effect on those elements. A 50-bp region in the vicinity of the grp78 TATA sequence is required for the OA stimulative effect of Tg induction. Interestingly, no sequence resemblance was found with known target sites of OA action (Wera et al., 1993; Kharbanda et al., 1993; Chauhan et al., 1994; Hyun et al., 1994), and the majority of the previously described OA response elements are upstream regulatory sequences. Thus, the grp78 target may represent a novel site. However, OA does not have to act directly on the transcription factors which bind to the 50 bp region. For example, it could inhibit a serine/threonine protein phosphatase activity which modifies the activity of any intermediatory regulatory components. Nonetheless, our finding that OA only exerts a modest effect on the Tg induction of grp78 and that the response does not involve the Tg response elements prompted us to investigate whether kinases other than that of the serine/threonine type are more critical elements for the Tg induction process in mammalian cells.
Our experiments indicate that genistein, a tyrosine
kinase inhibitor, almost totally eliminated the Tg induction response.
Furthermore, we have found that the grp core, the C3 and C1,
which are major targets for Tg induction, are also major targets of the
inhibitive effect exerted by genistein. Thus, genistein acts either
directly on the transcription factors which interact with these
promoter regulatory elements, or it modifies other targets in the
intermediary pathways leading to the activation of the nuclear
transcription factors. Recent examples suggest that genistein can exert
its effect both ways. In the T cell antigen receptor signaling system,
genistein was used to identify a tyrosine kinase activity which
down-regulates specifically calcium-mediated signaling at a point
downstream of the tyrosine kinase p56 but upstream of
calcium mobilization (Baldari and Telford, 1994). Genistein, on the
other hand, blocks the transcriptional stimulation of
interferon-induced genes by inhibiting tyrosine phosphorylation of the
transcription factor ISGF3 which binds directly to the
interferon-stimulated response element found in the promoter of
inducible genes (Gutch et al., 1992). In the grp78 promoter, the core, C3, and C1 act in concert to enhance grp expression (Wooden et al., 1991). Thus, it is possible
that upon stress induction, as in the case of ER Ca
depletion, a common tyrosine kinase activity is activated which
leads to the modification of the binding factors or their
co-activators, resulting in grp78 induction.
Among kinase
inhibitors, genistein is a specific inhibitor for tyrosine kinases but
not serine and threonine kinases and other ATP analogue-related enzymes
(Akiyama et al., 1987). Thus, it has minimal effect on the
enzyme activities of cAMP-dependent protein kinases, protein kinase C,
phosphorylase kinase, 5`-nucleotidase and phosphodiesterase. While it
is highly potent against tyrosine kinases such as the EGF receptor,
pp60 and pp110
, it is also capable of
inhibiting tyrosine kinases other than these three well characterized
ones. Since the targets for genistein can be broad, we initiated
experiments to determine which type of tyrosine kinase activity is
involved in the Tg induction of grp78. We found that treatment
of the cells with lavendustin-A, a specific inhibitor for EGF
receptor-associated tyrosine kinase, neither stimulates nor inhibits
the Tg response, suggesting that the tyrosine kinase activity mediating
the grp response to Tg is likely to be distinct from that of
the EGF receptor type. Based on our findings, we hypothesize that a
tyrosine kinase activity is essential for grp induction by Tg.
It is also possible that genistein inhibits grp induction
through a yet unknown mechanism. The identification of the genistein
sensitive components, their subcellular localizations and regulatory
mechanism will contribute significantly to the understanding of how ER
protein genes respond to Ca
depletion stress in
mammalian cells.