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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4379-4386
By
From the Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX; the Hematology Branch, National Heart, Lung
and Blood Institute, Bethesda, MD; and the Hematology-Oncology
Division, Brigham and Women's Hospital, Boston, MA.
The FAC protein encoded by the gene defective in Fanconi anemia (FA)
complementation group C binds to at least three ubiquitous cytoplasmic
proteins in vitro. We used here the complete coding sequence of
FAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a
B-lymphocyte cDNA library. Binding was confirmed by
coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear,
lysates of transfected COS-1 cells, as well as from mouse liver
cytoplasmic extracts. Deletion mutants of FAC showed that residues
103-308 were required for interaction with GRP94, and a natural
splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the
rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the
cellular phenotype of FA. Our results demonstrate that GRP94 interacts
with FAC both in vitro and in vivo and regulates its intracellular
level in a cell culture model. In addition, the pathogenicity of the
IVS-4 splicing mutation in the FAC gene may be mediated in part
by its inability to bind to GRP94.
THE AUTOSOMAL RECESSIVE disorder Fanconi
anemia (FA) frequently leads to bone marrow failure, leukemia, and
other cancers at an early age.1,2 Hence, it is regarded as
an important genetic model of preleukemia and cancer susceptibility.
Cellular manifestations of FA include chromosomal instability, enhanced sensitivity to bifunctional cross-linking agents (eg, mitomycin C
[MMC]) and to oxygen,3 cell cycle defects characterized
by delays in the G2 phase of the cell cycle (Seyschab et
al4 and the references therein) and a predisposition to
apoptosis.5-8 Although the molecular basis of these
abnormalities is not well understood, we and others have challenged
earlier views9 on the pathogenesis of FA and suggested that
it does not necessarily involve a primary defect in DNA
repair.10-12
At least eight distinct complementation groups of FA have been defined
by somatic cell fusions,13-16 three of which map to different chromosomal loci.13,17,18 The genes for
complementation groups A (FAA)19,20 and C
(FAC)21 have been cloned, and mutations consisting
of deletions, a splicing defect, as well as nonsense and missense
mutations have been identified (for examples, see Whitney et
al,22 Verlander et al,23 Gibson et
al,24 and Lo Ten Foe et al25). However, the
predicted polypeptide sequences of FAA and FAC have not shed light on
their biological functions. The 163-kD FAA protein contains a putative
nuclear localization signal,19,20 but otherwise it has
little homology to known proteins, including FAC. It may play a role in
DNA repair.26 Although fibroblasts from certain FA patients
have elevated rates of homologous recombination, the significance of
this observation for particular complementation groups and for the
proximal effects of FA proteins are unknown.27 The FAC
protein is considerably smaller (63 kD),10,11,21 and
cytoplasmic residence is obligatory for its ability to correct the
hypersensitivity of FA group C (FA-C) cells to bifunctional
alkylators.12
We have shown that at least three ubiquitous cytoplasmic proteins
interact with FAC.28 One of the interacting proteins is the
microsomal enzyme NADPH:cytochrome c (P-450) reductase. The amino-terminal region of FAC binds to the cytosolic, membrane-proximal domain of the reductase and attenuates its catalytic activity (H. Youssoufian et al, manuscript submitted). We postulate
that, in the absence of FAC, excess reactive metabolites of MMC and perhaps other compounds that are subject to bioreductive modification could damage DNA and compromise chromosomal stability.
In this study, we show that FAC binds directly to a member of the
glucose-regulated family of proteins, GRP94.29-31 This
abundant, stress-inducible glycoprotein with homology to the molecular
chaperone HSP90 is localized primarily in the lumen of the endoplasmic
reticulum (ER). There is also evidence for a transmembrane form of
GRP94 that spans the ER membrane and extends into the
cytoplasm.32-34 GRP94 has been shown to bind to both normal
and mutant proteins,32-36 including
p185erbB2 and a mutant form of herpes simplex
virus-1 glycoprotein B. GRP94 may act in tandem with GRP78 on partially
folded intermediates of the nascent Ig chains.35 It has
also been implicated in a variety of other cellular
processes,29 particularly intracellular Ca2+
homeostasis.37 This association with FAC establishes a
novel, albeit indirect, function for GRP94 in the maintenance of
chromosomal stability in mammalian cells.
Cell culture.
NRK cells stably transfected with GRP78-promoter constructs (gift of Dr
Amy Lee, University of Southern California, Los
Angeles)38 driving a GRP94 ribozyme (NRK-Ribo)
and a vector control (NRK-Vect) were maintained in Dulbecco's Modified
Essential Medium (DMEM; GIBCO-BRL, Grand Island, NY) containing 10%
fetal bovine serum (FBS) and G418 (400 µg/mL). COS-1, HSC536 (FA-C
lymphoblastoid cells), and HSC536-FAC (HSC536 cells transfected with
wild-type FAC) were maintained as described
previously.11,12
Plasmid constructions and cDNA libraries.
Mammalian expression vectors encoding wild-type FAC cDNA and a
fusion cDNA of FAC with the constant region of the IgG1 heavy chain (FAC-Ig) have been described previously.28 As a
control for FAC-Ig, seven residues from the amino-terminal region of
FAC were fused in-frame with the IgG1 epitope in the vector pED6 to generate Yeast two-hybrid screen.
The yeast strain HF7c was transformed initially with pGBT-FAC by the
lithium acetate procedure (MATCHMAKER Two Hybrid System; Clontech) and
grown on synthetic minimal medium (SD) in the absence of Trp. As
expected, colonies appearing on the plate lacked transcriptional activity. Next, pooled colonies were transformed with cDNA libraries (200 µg) and selected on SD plates in the presence of 50 fmol/L 3-aminotriazole, but lacking Leu, Trp, and His. Colonies that grew in
the absence of these amino acids were tested for their ability to
activate the lacZ reporter gene. Plasmids recovered from such
LacZ+ colonies were used to transform Escherichia
coli, which were selected on M9 plates in the absence of Leu to
obtain activation-domain plasmids.
Analysis of yeast two-hybrid inserts.
The inserts obtained from the yeast two-hybrid screen were
characterized by DNA sequencing using Sequenase Version 2.0 and analyzed by the BLAST program of the National Center for Biotechnology Information database.
Cell transfections.
COS-1 cells at 40% to 60% confluence in 100-mm dishes were
transfected with plasmid DNA at a concentration of 1 µg/mL by the diethylaminoethyl ether (DEAE)-dextran procedure as
before.28 Cells were then cultured in DMEM-10% FBS for 16 hours, detached with trypsin, and replated in fresh medium. NRK-Vect
and NRK-Ribo cells were transiently transfected with Lipofectamine
(GIBCO-BRL) for 5 hours with 20 µg of total plasmid DNA according to
the suggestions of the manufacturer. After 16 hours, 2 × 106 cells detached with trypsin were replated on 60-mm
dishes for ribozyme induction.
Immunoprecipitation and immunoblotting.
After 48 hours (for transfected COS-1 cells) or 72 hours (for
transiently transfected NRK cells), the monolayer was washed with cold
phosphate-buffered saline and detached by scraping, and
cytosolic extracts were prepared by incubation of the cell pellets in
hypotonic buffer (20 mmol/L HEPES, pH 7.9, 10 mmol/L NaCl, 10%
glycerol, 0.5 mmol/L EDTA, protease inhibitors). Nuclei were pelleted
by centrifugation at 600g for 5 minutes, and the supernatant
was centrifuged at 100,000g for 30 minutes to obtain a
cytosolic extract. Soluble liver extracts from C57BL/6 mice were
prepared by homogenization in ice-cold buffer (20 mmol/L HEPES, pH 7.9, 150 mmol/L NaCl) containing protease inhibitors. Nuclei and cellular
debris were pelleted by centrifugation at 600g for 10 minutes.
The final concentration of all of the extracts was adjusted to 20 mmol/L HEPES (pH 7.9), 50 mmol/L NaCl, 5% glycerol, 0.25 mmol/L EDTA,
and 0.1% Nonidet P-40. In some experiments, the concentration of NaCl
or EDTA was increased to 150 mmol/L and 2 mmol/L, respectively.
Extracts of transfected cells were incubated with the anti-FAC antibody
typically for 2 hours and with protein A-agarose beads for 1 hour at
4°C. Liver extracts were incubated with either anti-FAC antibody or
with a rat monoclonal antibody specific for GRP94 (StressGen
Biotechnologies Corp, Victoria, British Columbia, Canada), and
immunocomplexes were precipitated with a mixture of immobilized protein
A and protein G. Beads were washed three times with binding buffer over
30 minutes and boiled in 1× Laemmli buffer containing 200 mmol/L
dithiothreitol, and the proteins were resolved by denaturing sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to polyvinylidene difluoride
(PVDF) membranes (NEN, Boston, MA) by electroblotting. After blocking in TBST buffer (50 mmol/L Tris, pH 8.3, 150 mmol/L NaCl,
0.05% Tween-20) containing 5% nonfat milk, filters were incubated
sequentially with the primary antibodies (1 µg/mL) for 2 hours and
appropriate secondary antibodies conjugated to horseradish peroxidase
(HRP; GIBCO-BRL) for 45 minutes, which were then washed and visualized
by chemiluminescence (ECL; Dupont, Wilmington, DE).
Determination of factor VIII level.
COS-1 cells transfected with pMT2-factor VIII were incubated for 36 hours in DMEM-10% FBS containing aprotinin. The conditioned medium was
clarified of cellular debris by centrifugation at 600g for 5 minutes, and factor VIII antigen level was determined by enzyme-linked
immunosorbant assay using the monoclonal antibodies ESH2 (American
Diagnostica, Greenwich, CN) and factor VIII (Genetics Institute,
Cambridge, MA) for capture and detection, respectively. Antigen levels
were confirmed by the COATEST chromogenic assay (Chromogenix/DiaPharma,
Franklin, OH) using purified recombinant factor VIII as standard.
Determination of cell survival.
For measurements of cell survival, 2 × 105
exponentially growing cells were exposed to MMC (0 to 250 ng/mL; Sigma
Chemical Co, St Louis, MO) for 5 days, and viable cells were counted by trypan blue exclusion.
Identification of GRP94-FAC interaction by yeast two-hybrid assay.
To identify proteins that interact with FAC, we used the yeast
two-hybrid system,39 a genetic strategy that can identify in vivo protein-protein interactions by exploiting the ability of
fusion proteins to reconstitute a transcriptional activator. Plasmids
encoding FAC cDNA fused with the DNA-binding domain of GAL4
(pGBT-FAC) and libraries of cDNAs (human B-lymphocyte, bone marrow,
fetal liver, and HeLa cells) fused to the transcriptional activation
domain of GAL4 (in pACT) were used to cotransform HF7c yeast cells.
Double transformant colonies were screened and selected under His,
Trp-, and Leu-nutrient conditions. Viable colonies were
then assayed for LacZ expression. Using full-length FAC as bait, we identified 15 positive clones: 2 clones from the B-lymphocyte library, 3 from bone marrow, 4 from fetal liver, and 6 from HeLa cells.
Both clones from the B-lymphocyte library conferred FAC-dependent expression of LacZ in yeast cells
(Fig 1A). Sequence analysis of these clones
showed the full-length coding region of human GRP94 (a 782-amino acid
polypeptide with a 21-amino acid signal peptide), in-frame with the
GAL4 DNA-activation domain.
GRP94-binding domain of FAC.
To further delineate the amino acid sequences that are important for
the interaction of FAC with GRP94, we constructed carboxy-terminal truncation mutants of FAC and subcloned into pGBT9 to generate fusion
proteins with the DNA-binding domain of GAL4 (Fig 1A). Yeast cells were
cotransformed with FAC constructs in pGBT9 and full-length GRP94 in
pACT. Fusion polypeptides containing residues 1-467 or 1-308 of FAC
bound to GRP94, as indicated by the transcriptional activation of
LacZ. By contrast, a fusion protein containing FAC residues
1-103 did not bind GRP94. Thus, residues 103-308 of FAC interact with
GRP94.
FAC and GRP94 can be coimmunoprecipitated.
The endogenous expression of FAC in most mammalian cells is very
low.11 To look for an interaction between FAC and GRP94 in
mammalian cells and characterize binding parameters, we used two
complementary strategies. First, we overexpressed GRP94 in COS-1 cells
singly or by cotransfection with either wild-type human FAC or a
functional isoform fused to the heavy chain of human IgG1 (FAC-Ig).
After 48 hours, we performed sequential immunoprecipitation and
immunoblotting experiments. Because FAC is localized primarily in the
cytoplasm,10,11 we prepared cytoplasmic lysates from COS-1
cells transfected with pED6-FAC and immunoprecipitated with anti-FAC
antibodies and protein A-agarose. Immunocomplexes resolved by SDS-PAGE
were then immunoblotted and probed with a rat monoclonal antidody
specific for GRP94. Lysates containing FAC-Ig were immunoprecipitated directly with protein A-agarose. In both cases, the expected 94-kD GRP94 protein coimmunoprecipitated with FAC and FAC-Ig
(Fig 2A). Immunoblotting of the total
lysates showed abundant expression of the human GRP94 in cells
transfected with pcDNA3-GRP94. The anti-GRP94 antibody also
cross-reacted with a 90-kD species in both transfected and
nontransfected COS-1 cells, which probably represents the endogenous
monkey GRP94 (Fig 2B). FAC-GRP94 complexes were not detected in cells
transfected with the empty vector, with GRP94 alone, or with
Modulation of intracellular levels of FAC with GRP94.
Does the interaction of FAC with GRP94 have any functional consequence?
To assess this possibility, we took advantage of a cell line in which
the expression of GRP94 can be conditionally inhibited by a targeted
ribozyme that can cleave the GRP94 mRNA.38 The ribozyme is
driven by the stress-inducible GRP78 promoter. Under steady-state
conditions, the activity of the promoter is minimal; consequently, the
expression of the ribozyme is also limited. However, under stress
conditions, the activation of the promoter can induce high levels of
the ribozyme, which degrades the GRP94 mRNA. Rat NRK cells stably
expressing GRP78 promoter constructs were transiently transfected with
pED6-FAC. After 48 hours, cells were either untreated (steady-state) or
treated with 100 nmol/L MMC for 24 hours (stress), and total cell
lysates were assayed for GRP94 and FAC expression simultaneously.
Lysates of NRK-Vect cells probed simultaneously for GRP94 and FAC
(Fig 4A) and subsequently for
GRP94 inactivation in vivo.
We also performed experiments to discount the possibility that reduced
level of GRP94 in ribozyme-activated cells results from a spurious
effect on vector-encoded cDNA. First, Northern analysis of cytoplasmic
RNA from NRK-Ribo cells transfected with pED6-FAC showed no differences
in the steady-state levels of FAC mRNA in MMC-treated or untreated
cells (data not shown). Thus, a transcriptional effect on FAC
expression would appear to be unlikely. Second, we identified another
molecular target of GRP94 that is regulated by protein-protein
interactions and analyzed its expression concomittantly with FAC. We
reasoned that such an independent assay for the intracellular function
of GRP94 could further minimize the possibility of an artifact caused
by MMC or the ribozyme on the observed variations in the levels of FAC. Previous studies have shown that secretion of the human coagulation protein factor VIII from transfected Chinese Hamster Kidney cells is
enhanced by antisense inhibition of GRP78.41 Because GRP78 and GRP94 can work in tandem,35 we tested the effect of
GRP94 levels on factor VIII secretion. In pilot studies, we
cotransfected pcDNA3-GRP94 and pMT2-factor VIII in COS-1 cells and
noted a significant reduction in the secretion of factor VIII with
GRP94 coexpression (data not shown). Next, we cotransfected NRK-Vect
and NRK-Ribo cells with pED6-FAC and pMT2-factor VIII. It should be
noted that pED6 and pMT2 are closely related vectors, and any potential
effect of the ribozyme on the vector is most likely comparable. After 24 hours, cells were exposed to 100 nmol/L MMC continuously for 36 hours and the conditioned media was assayed for factor VIII activity.
There was a significant increase in factor VIII activity in the media
of NRK-Ribo cells treated with MMC, but not in NRK-Vect cells
(Fig 5A). Third, an alternative possibility
is that FAC regulates the intracellular level of GRP94. In this case,
the level of GRP94 should be different in parental FA-C cells compared with complemented cells. Immunoblot analysis of total cellular lysates
from HSC536 and HSC536-FAC cells showed no significant variations in
the level of GRP94 (Fig 5B). There was also little variation in GRP94
levels in cells of other FA groups and in non-FA cells (data not
shown). Thus, the alternative possibility that FAC regulates the
expression of GRP94 is not supported by these data.
GRP94 inactivation causes hypersensitivity to MMC.
The hallmark of FA cells is their hypersensitivity to bifunctional
cross-linking agents. By conditional inactivation of FAC, we reasoned
that NRK-Ribo cells should display an enhanced sensitivity to MMC.
NRK-Vect and NRK-Ribo cells were treated with MMC and viable cells were
counted after 5 days. The results of two separate experiments are shown
(Fig 6). NRK-Ribo cells were significantly more sensitive to the cytotoxic effects of MMC than NRK-Vect cells. The
difference in the absolute values between the two experiments may
reflect unequal levels of ribozyme activation by MMC. These results
demonstrate that inactivation of GRP94 leads to a
cross-linker-sensitive phenotype similar to that of FA cells.
In this study, we demonstrate that FAC-GRP94 immunocomplexes can be
detected in mammalian cells, and residues 103-308 of FAC are essential
for this interaction. At first glance, the abundance of heat-shock
factors and their frequent isolation from yeast two-hybrid screens,
possibly through interactions with misfolded domains, might raise
concern that the interaction of FAC with GRP94 is of dubious biological
significance. However, both structural mapping studies and functional
evidence show that, in this instance, this possibility is unlikely.
First, we demonstrate that GRP94 can form protein complexes in normal
mouse liver cells. Relatively large amounts of extract were required
for this experiment, because the expression of both human FAC and
murine fac is low in most mammalian cells. Our ability to demonstrate
such an interaction in normal cells gives credence to the data from
heterologous systems. Second, the identification of a specific binding
domain on FAC makes it less likely that the observed interaction
results are artifactual. FAC-GRP94 complexes are stable at relatively
low salt concentrations, but dissociate in both higher salt
concentrations and in the presence of EDTA. These observations suggest
that the interaction of FAC with GRP94 may be transient or of
low-affinity and subject to regulation by divalent cations. Third, our
preliminary experiments fail to show an interaction between GRP94 and
FAA (H. Youssoufian, unpublished observation, November
1997), which also contains a large number of leucine
residues and other hydrophobic segments. Fourth, and most important,
the use of a powerful genetic system in mammalian cells demonstrates
that the FAC-GRP94 biochemical interactions are functionally relevant.
Ribozyme-mediated inactivation of GRP94 reduces the level of GRP94
protein, which in turn greatly reduces the level of immunoreactive FAC.
Concomittant with these reductions is an acquired sensitivity of the
host cells to MMC. Taken together, our data support the conclusion that
GRP94 most likely interacts with FAC in vivo and regulates its
intracellular level. A plausible interpretation might be that GRP94
prevents the premature degradation of FAC.
Submitted September 5, 1997;
accepted January 27, 1998.
The authors thank Drs Amy Lee and Randall J. Kaufman for their gifts of
cell lines and reagents, Sally Tetrault for technical assistance, and
Dr Neal Young for stimulating discussions.
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Abstract
Introduction
Abstract
FAC-Ig. Full-length FAC cDNA was also fused
in-frame downstream of the DNA-binding domain of GAL4 in the vector
pGBT (Clontech, Palo Alto, CA) to generate pGBT-FAC. Carboxy-terminal deletion mutants of FAC and an interstitial deletion that
removes exon 4 (the product of the IVS4+4 mutation, which can generate a 111-bp, in-frame deletion of exon 4)
-Galactosidase activity.
Yeast transformants were assayed for 
, white color after 12 hours; +,
blue color after 1 to 12 hours; ++, dark blue color or blue color
after less than 1 hour.
.
B.