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Molecular and Cellular Biology, July 2000, p. 4724-4735, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Fanconi Anemia Protein FANCC Binds to and
Facilitates the Activation of STAT1 by Gamma Interferon and
Hematopoietic Growth Factors
Qishen
Pang,1,2
Sara
Fagerlie,1
Tracy A.
Christianson,2
Winifred
Keeble,2
Greg
Faulkner,2
Jane
Diaz,2
R. Keaney
Rathbun,2 and
Grover
C.
Bagby1,2,*
Oregon Cancer Center, Department of Medicine
(Division of Hematology and Medical Oncology) and Department of
Molecular and Medical Genetics, Oregon Health Sciences
University,1 and Molecular
Hematopoiesis Laboratory, VA Medical Center,2
Portland, Oregon 97201
Received 14 September 1999/Returned for modification 24 February
2000/Accepted 7 April 2000
 |
ABSTRACT |
Hematopoietic progenitor cells from Fanconi anemia (FA) group C
(FA-C) patients display hypersensitivity to the apoptotic effects of
gamma interferon (IFN-
) and constitutively express a variety of
IFN-dependent genes. Paradoxically, however, STAT1 activation is
suppressed in IFN-stimulated FA cells, an abnormality corrected by
transduction of normal FANCC cDNA. We therefore sought to define
the specific role of FANCC protein in signal transduction through
receptors that activate STAT1. Expression and phosphorylation of
IFN-
receptor
chain (IFN-
R
) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in
coimmunoprecipitation experiments STAT1 did not dock at the IFN-
R of
FA-C cells, an abnormality corrected by transduction of the FANCC
gene. In addition, glutathione S-transferase fusion genes
encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of
IFN-
-stimulated B cells and IFN-, granulocyte-macrophage
colony-stimulating factor- and stem cell factor-stimulated MO7e cells.
Kinetic studies revealed that the initial binding of FANCC was to
nonphosphorylated STAT1 but that subsequently the complex moved to the
receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and
STAT1-DNA complexes were not detected in nuclear extracts of FA-C
cells. We also determined that the IFN-
hypersensitivity of FA-C
hematopoietic progenitor cells does not derive from STAT1 activation
defects because granulocyte-macrophage CFU and erythroid burst-forming
units from STAT1
/
mice were resistant to IFN-
.
However, BFU-E responses to SCF and erythropoietin were suppressed in
STAT
/
mice. Consequently, because the FANCC protein
is involved in the activation of STAT1 through receptors for at least
three hematopoietic growth and survival factor molecules, we reason
that FA-C hematopoietic cells are excessively apoptotic because of an
imbalance between survival cues (owing to a failure of STAT1 activation
in FA-C cells) and apoptotic and mitogenic inhibitory cues
(constitutively activated in FA-C cells in a STAT1-independent fashion).
 |
INTRODUCTION |
Gamma interferon (IFN-
) induces
transcription of a distinct set of genes by activating STAT1, one
member of a family of latent cytoplasmic transcription factors that are
activated via phosphorylation on tyrosine residues (6). The
IFN-
receptor (IFN-
R) lacks intrinsic tyrosine kinase activity,
but on ligand binding, receptor multimerization results in reciprocal
activation of JAK1 and JAK2, two Janus tyrosine kinases noncovalently
attached to the IFN-
R
and
chains (2, 20, 22).
Phosphorylation of the tyrosine residue on IFN-
R
by JAK molecules
creates a docking site for the Src homology 2 domain of STAT1, which is
then phosphorylated on tyrosine and ultimately forms a homodimeric
transcription factor that translocates to the nucleus (6,
21). The factors that govern the traffic of cytoplasmic STAT
molecules to the docking site on the IFN-
R are unknown. We have
recently found that the activation of STAT1 in response to IFN-
is
suppressed in hematopoietic cells from children with Fanconi anemia of
type C (FA-C) and in mice nullizygous at the FA-C locus. However, in
the ground state (uninduced by IFN), IFN response factor 1 (IRF-1) is
expressed at high levels in mutant FA-C cells (35),
suggesting that a non-STAT1 pathway is involved in constitutive
activation of IRF-1 in FA cells. In addition, complementation of the
defect by retrovirus mediated transfer of normal FANCC cDNA
reconstitutes the normal STAT1 response (10, 38).
Linkage of FANCC function with that of STAT1 provided us with an
opportunity to test whether the relationship of these two molecules was
direct or indirect. We report herein results of experiments in which
the assembly of the fully activated IFN-
R complex, including STAT1,
JAK1, and JAK2, was examined in isogenic murine and human FA-C cells.
We report that in IFN-
-stimulated FA-C cells, phosphorylation of
JAK1, JAK2, and IFN-
R
occurs normally, but STAT1 does not dock at
the receptor
chain. In FA-C cells nuclear STAT1 is reduced, and IFN
fails to induce STAT1-specific DNA-binding complexes and expression of
IRF-1. Expression of the normal FANCC cDNA in mutant cells results
in normal STAT1 docking and phosphorylation as well as normal induction
of nuclear STAT1-DNA complex and normal induction of IRF-1. We also
find that a variety of cytokines and hematopoietic growth factors
stimulate the association of STAT1 with glutathione
S-transferase (GST) fusion proteins encoding the normal
FANCC but not a naturally occurring inactivating mutant FANCC
(L554P) and that the association occurs rapidly and prior to STAT1
phosphorylation on Y701. Coimmunoprecipitation experiments
confirmed the IFN-inducible association of endogenous FANCC and
STAT1 in normal but not mutant cells. We propose that FANCC is a
control factor for STAT1 traffic to receptor docking sites, and that
mutations that inactivate FANCC function interdict this function.
We propose that the hematopoietic defects in FA derive, at least in
part, from a loss of balance between mitogenic cues (owing to a
reduction of signals transduced through receptors for growth factors
that activate STAT1) and mitogenic inhibitory cues (secondary to
FANCC-dependent, STAT1-independent constitutive activation of
mitotic inhibitory factors), in particular IRF-1, a factor known to
effect apoptotic responses in hematopoietic progenitor cells
(43).
 |
MATERIALS AND METHODS |
Cell cultures and IFN-
stimulation.
Epstein-Barr
virus-transformed lymphoblasts were maintained in RPMI 1640 medium
(RPMI; Life Technologies, Grand Island, N.Y.) supplemented with 15%
heat-inactivated fetal calf serum and grown in a humidified 5%
CO2-containing atmosphere at 37°C. The lymphoblast lines
JY and HSC536N (FA-C) have been described elsewhere (38). Unless otherwise indicated, cells were stimulated with recombinant human IFN-
(1 ng/ml; R & D Systems, Minneapolis, Minn.) for the indicated time periods. For detection of STAT1 phosphorylation, cells
were serum starved for 2 h prior to IFN-
treatment. The leukemia cell line MO7e and its high erythropoietin receptor
(EPOR)-expressing subline MO7ER (51) were maintained in RPMI
containing 20% fetal calf serum and granulocyte-macrophage
colony-stimulating factor (GM-CSF; 100 U/ml; Genetics Institute,
Cambridge, Mass.) or EPO (2 U/ml; Amgen Inc., Thousand Oaks, Calif.).
Before stimulation, cells were washed and incubated for 18 h at
37°C in serum-free RPMI containing 0.5% bovine serum albumin (Sigma
Chemical Co., St. Louis, Mo.). After starvation, cells were washed and
then exposed to IFN-
(10 ng/ml), IFN-
(20,000 U/ml; Schering
Corp., Kenilworth, N.J.), GM-CSF (100 U/ml), SCF (50 ng/ml; R & D
Systems), and EPO (40 U/ml) for 10 min.
Retroviral infection and transduction of FA cell lines.
FANCC cDNA (47) was subcloned into the retroviral vector
pLXSN as described previously (38). Plasmid pLXSN-FANCC
(10 µg) was transfected by the calcium phosphate precipitation method into PA12 producer cells expressing the vesicular stomatitis virus G
envelope protein (26). Retroviral supernatants were
collected and used for transduction of FA-C cell lymphoblasts. HSC536N
cells were exposed to the filtered supernatants containing virus in the
presence of Polybrene (8 µg/ml; Sigma). Sets of isogenic lines were
selected in G418 and then exposed to multiple doses of mitomycin C in
cytotoxicity (trypan blue viability) and apoptosis (TUNEL [terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling]
and annexin V binding) assays to ensure that the mutant cells
transduced with the normal cDNA were fully complemented. HSC5326N/FANCC
cells are HS536N cells transduced by the FANCE vector. HSC536N/neo
cells are HS536N cells transduced by the vector alone.
Preparation of whole-cell lysates (WCL).
Following treatment
with IFN-
, cells were washed twice with ice-cold phosphate-buffered
saline (PBS) and lysed with either radioimmunoprecipitation assay
(RIPA) buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.6], 1% sodium
deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS])
or Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 20 mM Tris-HCl [pH
8.0], 137 mM NaCl, 10% glycerol). Both lysis buffers were
supplemented with the following freshly made protease inhibitors: 1%
aprotinin, 1 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 2 mM sodium orthovanadate. For cell lysates used
specifically for assessment of protein-protein interactions, cells were
lysed in digitonin lysis buffer (1% digitonin, 50 mM Tris-HCl [pH
8.0], 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate) containing
the protease inhibitors aprotinin, leupeptin, and PMSF. After rocking
at 4°C for 20 min (60 min when cells were lysed with digitonin
buffer), cell lysates were cleared by centrifugation at 13,200 rpm for 20 min at 4°C, and protein concentrations were determined by the Bradford method (4) using a protein microassay reagent
(Bio-Rad, Hercules, Calif.).
Electromobility shift assay (EMSA).
Nuclear extracts were
prepared by the method of Dignam et al. (8). A human IRF-1
IFN-
activation sequence (GAS) oligonucleotide (5'-ACAACAGCCTGATTTCCCCGAA-3') was synthesized in the
Molecular Biology Core Lab (Portland VA Medical Center) and then
labeled with [
-32P]ATP to 2.5 × 104
cpm/ng, using T4 polynucleotide kinase (Boehringer Mannheim). The
binding reaction mixture (20 µl) contained 5.0 to 7.5 µg of nuclear
extracts, 0.2 ng of labeled oligonucleotide 2 µg of poly(dl-dC), and
10 µg of bovine serum albumin in 10 mM Tris-Cl (pH 7.4)-50 mM
NaCl-1 mM dithiothreitol (DTT)-1 mM EDTA-10% glycerol. Reaction mixtures were incubated at room temperature for 30 min and then resolved on a 4% polyacrylamide gel in 25 mM Tris, 190 mM glycine, and
1 mM EDTA. Gels were dried and autoradiographed with intensifying screens at
80°C.
Reverse transcription-PCR.
Total RNA was isolated from cells
using Tri Reagent (Molecular Research Center, Inc., Cincinnati, Ohio)
in accordance with the manufacturer's instructions. First-strand cDNA
was reverse transcribed from the indicated RNA using random
hexanucleotide primers (Gibco BRL) and mouse mammary tumor virus RNase
H
reverse transcriptase (Gibco BRL) as previously
described (46). The cDNA was then amplified by PCR for 35 cycles (denatured at 94°C for 30 s, primer annealed at 53°C
for 30 s, and primer extended at 72°C for 30 s). Control
reactions in which no cDNA (i.e., water only) was added to the PCR
mixture were analyzed with each experiment. For IRF-1 amplification,
the primers used were 5'-GAGCTGGGCCATTCACACAG-3' and
5'-CATGGCGACAGTGCTGGAGT-3', which produced an amplimer with a predicted length of 390 nucleotides. The amplification products were
separated by electrophoresis on 1.5% agarose, stained with ethidium
bromide, and visualized by UV transillumination.
Antibodies.
Antibodies used included antiphosphotyrosine
(anti-P-Tyr) antibody 4G10, provided by Brian Druker (Oregon Health
Sciences University, Portland), anti-JAK1 monoclonal antiserum
(Transduction Laboratories, Lexington, Ky.), anti-JAK2 polyclonal
antiserum (Upstate Biotechnology, New York, N.Y.), anti-IFN-
R
polyclonal antibody (Antigenix America Antibodies, New York, N.Y.),
anti-STAT1
monoclonal antiserum from Santa Cruz Biotechnology (Santa
Cruz, Calif.), rabbit polyclonal anti-Stat3, anti-Stat5, and antibodies specific for the tyrosine-phosphorylated forms of STAT1 (P-STAT1), Stat3, and Stat5 (New England Biolabs, Beverly, Mass.), anti-FANCC polyclonal antiserum as described previously (52), and sheep anti-mouse and donkey anti-rabbit horseradish peroxidase-conjugated antibody (Amersham, Arlington Heights, Ill.). All antibodies were used
in accordance with the manufacturer's instructions.
Immunoprecipitation.
WCLs (about 1 mg of total proteins)
were precleared with 50 µl of 50% protein A-Sepharose suspension
(Pharmacia Biotech, Inc.) for 1 h at 4°C. After separation of
the protein A-Sepharose from the lysate by centrifugation at 1,000 rpm
for 1 min at 4°C, the lysate was incubated with either anti-P-Tyr
antibody 4G10 (12 µg/ml), anti-IFN-
R
(2 µg/ml), or anti-STAT1
(1 µg/ml) for 3 to 5 h at 4°C with constant agitation.
Immunocomplexes were then bound to protein A-Sepharose beads (50 µl
of 50% slurry) during a 1- to 2-h incubation at 4°C. The
immunoprecipitates were recovered by centrifuging at 1,000 rpm for 1 min at 4°C. If cells were lysed in NP-40 lysis buffer, the
immunocomplexes were washed three times with 0.05% NP-40 wash buffer
(0.05% NP-40 in 1× PBS). If cells were lysed in digitonin lysis
buffer, the immunocomplexes were then washed with 0.1% digitonin wash
buffer (same as digitonin lysis buffer except that the concentration of
digitonin was 0.1%).
Immunoblotting.
WCL, nuclear extracts, or immunocomplexes
were prepared as above and subjected to immunoblot analyses. Samples
were heated at 94°C for 5 min in 2× Laemmli SDS sample buffer,
separated by SDS-polyacrylamide gel electrophoresis (PAGE), and
transferred onto Bio-Blot nitrocellulose membranes (Costar, Cambridge,
Mass.). Nonspecific binding sites on the membranes were blocked with
Tris-buffered saline plus 0.05% Tween 20 containing 5% nonfat dry
milk for 1 h at room temperature or overnight at 4°C.
Immunoblots were subsequently incubated with the indicated primary
antibodies at a dilution according to the manufacturer's instruction.
After stringent washing, the blots were incubated with appropriate
secondary antibodies (linked to horseradish peroxidase) for 30 min at
room temperature and developed by using an enhanced chemiluminescence
kit (Amersham).
GST-FANCC fusion protein purification and in vitro binding
assays.
The GST-FANCC and GST-L554P fusion constructs were
created by insertion of the entire open reading frame of FA-C and its
mutant version, L554P, into the BamHI and SmaI
sites of yeast overexpression vector pGST (31). The plasmids
were transformed into yeast strain Sc334 (19) using a
Frozen-EZ yeast transformation kit (ZYMO Research, Orange, Calif.).
Expression and purification of the yeast GST fusion proteins were
performed as described elsewhere (31, 32), with minor
modifications. The GST fusion protein was prepared by suspending the
yeast cells containing pGST, pGST-FANCC, or pGST-L554P in an equal
volume of buffer A (37.5 mM K2PO4 [pH 9.5],
7.5 mM DTT, 1.5 mM EDTA, 1 M NaCl, 2.5% glycerol, 7.5% ethylene glycol, 0.03% silicon antifoam), and the cells were broken by vortexing with an equal volume of glass beads (eight 15-s bursts). The
lysate was removed from the beads, and Triton X-100 was added to 1%.
After being clarified twice by centrifugation, the supernatant was
incubated for 30 min at 4°C with 0.1 ml of glutathione-Sepharose 4B
(Pharmacia Biotech, Piscataway, N.J.) 50% slurry equilibrated with
buffer B (50 mM KPO4 [pH 7.2], 1 mM DTT, 0.5 mM EDTA, 1 M NaCl, 10% glycerol, 1% Triton X-100). The Sepharose beads were washed
three times with buffer B containing 0.5 M NaCl. For binding, about 10 µg of GST, GST-FANCC, or GST-L554P fusion protein bound to
glutathione-Sepharose beads was incubated with WCL from untreated or
IFN-
-treated JY cells (1 mg of total proteins) prepared in 1%
digitonin buffer supplemented with 1% aprotinin, 1 µg of leupeptin per ml, 1 mM PMSF, and 2 mM sodium orthovanadate. The final
concentration of digitonin in the binding mixture was adjusted to
0.5%. After shaking at 4°C for 40 min, the beads were recovered and
washed three times with 0.1% digitonin buffer before boiling in
Laemmli SDS sample buffer and immunoblotting as described above. For
WCL input controls, 60 to 100 µg of total WCL proteins was loaded in
each lane.
In vivo phosphate labeling.
Cells were starved for 90 min in
phosphate-free RPMI containing 15% dialyzed fetal bovine serum, and
[32P]orthophosphate (150 µCi/ml; DuPont NEN) was added
to the medium. After labeling for 3 h, cells were incubated with
IFN-
(10 ng/ml) for the indicated time points. WCL prepared as
described above were subjected to affinity precipitation with
GST-FANCC and analyzed by SDS-PAGE followed by autoradiography.
Western blot analysis was performed on these precipitates to determine
the quantity of both tyrosine-phosphorylated and total STAT1 bound to
GST-FANCC.
Murine clonal BM cell cultures.
Bone marrow (BM) samples
were obtained from an equal number of FANCC
/
and
FANCC
/+ mice (55) and
STAT
/
and STAT+/
mice (9, 28)
for each experiment. Femoral marrow samples obtained from mice after
cervical dislocation were washed and resuspended in RPMI medium for
viable counts (trypan blue dye exclusion). Unfractionated BM cells
(105) were cultured in 1 ml of methoCult H4230 (Stem Cell
Technologies, Vancouver, British Columbia, Canada),
penicillin-streptomycin (Life Technologies), and either murine SCF
(from 0.10 to 10 ng/ml; R & D Systems), murine EPO (2 ng/ml), or both
SCF and EPO. A combination of three growth factors was used as a
positive control: murine Steel factor (10 ng/ml; R & D Systems), murine
interleukin-3 (IL-3; 10 ng/ml; R & D Systems), and human EPO (2 U/ml;
Amgen). Granulocyte-macrophage CFU (CFU-GM) were cultured in
35-mm-diameter tissue culture dishes at 37°C in 5% CO2
in air and counted after 7 and 14 days, using a dissecting microscope.
Colony growth results were expressed as mean (of triplicate plates)
colonies per plate ± standard deviation (SD). Between-group
comparisons were made using one-way analysis of variance.
 |
RESULTS |
Deficient STAT1
activation in FA-C cells.
We have recently
shown that IFN-
-induced STAT1 tyrosine phosphorylation is reduced in
FA-C cells (38). Equal numbers of cells from each isogenic
line were serum starved for 2 h followed by stimulation with
IFN-
at 37°C for the indicated time periods. WCL were prepared in
RIPA buffer (see Materials and Methods) and analyzed by immunoblotting
with antisera specific to P-STAT1. As shown in Fig.
1, the FA-C mutant cell lines HSC536N and
HSC536N/neo contained less P-STAT1 than the normal B-cell line JY
following IFN-
stimulation (Fig. 1A, lane 2 versus lanes 4 and 8).
Introduction of the normal FANCC gene into FA-C cells
(HSC536N/FANCC) corrected the STAT1 activation defect (Fig. 1A,
lane 6), suggesting that STAT1 activation via tyrosine phosphorylation
is dependent on expression of a normal FANCC gene product.
Reprobing the blot with anti-STAT1 antibody showed that STAT1 protein
was expressed at comparable levels in all four cell lines, as indicated
in the lower panel.

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FIG. 1.
Defective STAT1 tyrosine phosphorylation in IFN
-stimulated FA-C cells. (A) Cells from the indicated FA-C isogenic
lines were serum starved for 2 h, incubated with IFN- for the
indicated time periods at 37°C, and then solubilized in RIPA lysis
buffer as described in Materials and Methods. The WCL were separated by
SDS-PAGE and blotted with antibodies to P-STAT1. (B) The blot described
above, stripped and reprobed with anti-STAT1. Here and in subsequent
figures, the label "FAC" represents FANCC, and the positions of
migration of protein standards (in kilodaltons) are indicated on the
left. (B) EMSA of STAT1 binding to oligonucleotides corresponding to
the STAT1-specific GAS element in the IRF-1 promoter. Nuclear extracts
were prepared from HSC536N/FANCC (lanes 1 to 4) and HSC536N (lanes
5 to 8) cells untreated (lanes 1 and 5) or treated with 1 ng of IFN-
per ml (lanes 2 to 4 and 6 to 8). Arrow denotes STAT1-GAS complexes.
(C) IFN- -induced expression of IRF-1 transcript. Horizontal rows
represent amplification from individual cell lines as indicated.
Vertical columns show samples from untreated cells at time zero (lane
1), untreated cells at 3 h (lane 2), cells treated with IFN-
(0.5 ng/ml) for 3 h (lane 3), and cells treated with IFN- (50 ng/ml) for 3 h (lane 4).
|
|
To ensure that FA-C cells had reduced STAT1 function, we sought to
identify nuclear STAT1 and found none at any time point
from 5 to 120 min after IFN-

stimulation (data not shown). Using
an EMSA with a
STAT1-specific oligonucleotide as a probe, we observed
STAT1-DNA
complexes in the nuclear extracts from IFN-stimulated
HSC536/FANCC
cells (Fig.
1B, lanes 1 to 4). In contrast, no constitutive
or
inducible STAT1-specific DNA-binding activity was detected
in the
nuclear extracts from mutant FA-C cells (Fig.
1B, lanes
5 to 8). In
addition, IFN-

induction of IRF-1 was also abolished
in mutant cells
(Fig.
1C) as we have described elsewhere (
38).
Tyrosine phosphorylation of IFN-
R
in IFN-
-stimulated B
lymphocytes.
Cellular responsiveness to IFN-
requires the
activation of IFN-
R
through phosphorylation on tyrosine residues
(12, 13, 22). We therefore asked whether ligand-induced
IFN-
R
tyrosine phosphorylation was different quantitatively and
temporally in FA-C cells. Cells were treated with IFN-
for the
indicated time, and lysates were incubated with anti-IFN-
R
antibodies. In the absence of IFN-
, no tyrosine-phosphorylated
IFN-
R
was detected in any of the isogenic cells (Fig.
2A, lanes 1, 4, 7, and 10). Following
addition of ligand, equivalent IFN-
R
tyrosine phosphorylation was
observed in all four isogenic cell lines (Fig. 2A, lanes 2, 3, 5, 6, 8, 9, 11, and 12). The kinetics of induced IFN-
R
tyrosine phosphorylation, which was maximal at 5 min and remained at maximal levels within 10 min of exposure to IFN-
, was similar in all four
cell lines. The amounts of the receptor proteins loaded in each lane
were comparable, as determined by reprobing the membrane with
anti-IFN-
R
antibody (Fig. 2B).

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FIG. 2.
Activation of IFN- R in response to IFN- in FA-C
cells. Cells were incubated with IFN- for the indicated time periods
at 37°C and then solubilized in NP-40 lysis buffer as described in
Materials and Methods. The WCL were immunoprecipitated (IP) with
anti-IFN- R antibody. The immunoprecipitates were then analyzed by
immunoblotting. (A) Cell lysates prepared from four FA-C isogenic lines
were immunoprecipitated with anti-IFN- R and probed with
anti-P-Tyr; (B) the blot described above was stripped and reprobed with
anti-IFN- R .
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|
Tyrosine phosphorylation of JAK1 and JAK2 kinases in FA-C
cells.
JAK1 and JAK2 are activated by IFN-
through tyrosine
phosphorylation, and these activated kinases play an essential role in
transduction of the IFN-
signal, at least in part, by
phosphorylating STAT1 (25, 30, 33, 35, 39, 54). To determine
whether IFN-
-induced tyrosine phosphorylation of the JAK kinases was normal in FA-C cells, WCL prepared from normal and FA-C cells were
first immunoprecipitated with anti-P-Tyr antibody 4G10 and then
immunoblotted with antibodies against JAK1 and JAK2. As shown in Fig.
3, tyrosine phosphorylation of JAK1 (Fig.
3A) and JAK2 (Fig. 3C) was evident in all four isogenic cell lines
after 5 min of IFN-
treatment. Tyrosine phosphorylation of JAK1 or
JAK2 was not detected in unstimulated cells (Fig. 3A and C, lanes 1, 3, 5, and 7). The kinetics of induced JAK phosphorylation were comparable
in normal and FA-C cell lines. To ensure that expression of JAK1 and
JAK2 proteins was equivalent in both normal and FA-C cells, we
performed immunoblotting with anti-JAK1 and -JAK2 antibodies, using WCL
from the same cell lines. As expected, both the untreated and
IFN-
-treated samples prepared from each cell line were found to
contain similar levels of JAK1 and JAK2 (Fig. 3B and D). These results
indicate that after ligation of IFN-
to its receptor, activation, by
tyrosine phosphorylation, of JAK1 and JAK2 was normal in FA-C cells.

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FIG. 3.
IFN- -induced tyrosine phosphorylation of JAK1 and
JAK2 in FA-C cells. Cells were stimulated with IFN- for various time
periods and lysed in NP-40 lysis buffer. The WCL were either directly
analyzed by immunoblotting with anti-JAK1 and -JAK2 antibodies or
immunoprecipitated (IP) with anti-P-Tyr antibody, and the
immunoprecipitates were then analyzed by immunoblotting. (A) Cell
lysates prepared from four FA-C isogenic lines were immunoprecipitated
with anti-P-Tyr and probed with anti-JAK1 antibody; (B) cell lysates
were directly immunoblotted with anti-JAK1 antibody; (C) cell lysates
prepared from FA-C isogenic lines were immunoprecipitated with
anti-P-Tyr and probed with anti-JAK2 antibody; (D) cell lysates were
directly immunoblotted with anti-JAK2 antibody.
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IFN-
-induced coimmunoprecipitation of IFN-
R
with STAT1
requires the FANCC protein.
IFN-
-induced tyrosine
phosphorylation of IFN-
R
creates a docking site for latent STAT1,
which, after docking, is then activated by the JAK kinases (6, 18,
22, 58). In support of this model, it has been shown that STAT1
associates with tyrosine-phosphorylated IFN-
R
both in vitro and
in vivo (12, 13, 42). Having established the normal pattern
of IFN-
-induced tyrosine phosphorylation of IFN-
R
and the two
JAK kinases in FA-C cells, we next asked whether STAT1 was recruited to
the activated IFN-
R complex. INF-
-treated isogenic cells were
solubilized with 1% digitonin buffer, and protein complexes were
immunoprecipitated using antibody to IFN-
R
. The presence of STAT1
in anti-IFN-
R
immunoprecipitates was assessed by immunoblotting
with either anti-P-Tyr or anti-STAT1
monoclonal antibody. When
material precipitated using lysates from FA-C isogenic cell lines was
analyzed with the anti-P-Tyr antibody, a diffusely migrating
phosphoprotein of ~100 kDa was detected in all four cell lines only
when cells had been treated with IFN-
(Fig.
4A, lanes 2, 4, 6, and 8). This 100-kDa
phosphoprotein was identified by reprobing the blot with
anti-IFN-
R
(Fig. 4E). In addition to the tyrosine-phosphorylated
IFN-
R
, a distinct phosphoprotein of ~90 kDa recognized by
anti-P-Tyr immunoblotting was also precipitated by anti-IFN-
R
in
lysates prepared from IFN-
-treated normal (JY) and complemented FA-C
(HSC536N/FANCC) cell lines but not in those from stimulated FA-C
(HSC536N and HSC536N/neo) cell lines (Fig. 4A, lanes 2 and 6 versus 4 and 8). Reprobing the blot with a monoclonal antibody specific to
STAT1
revealed that the 90-kDa band was STAT1
(Fig. 4C, lanes 2 and 6). No signal was detected in the anti-STAT1
immunoblot when
anti-IFN-
R
immunoprecipitates had been prepared from
IFN-
-treated mutant cell lines HSC536N and HSC536N/neo (Fig. 4C,
lanes 4 and 8) or from cells that had not been incubated with IFN-
(Fig. 4C, lanes 1, 3, 5, and 7).

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FIG. 4.
IFN- -induced formation of STAT1 -IFN- R
complex in B lymphocytes. Cells were stimulated with IFN- for up to
10 min and solubilized in digitonin lysis buffer as described in
Materials and Methods. The WCL were then incubated with
anti-IFN- R or anti-STAT1 antibody. The immunoprecipitates were
analyzed by SDS-PAGE followed by immunoblotting. (A) Cell lysates
prepared from FA-C isogenic lines were immunoprecipitated (IP) with
anti-IFN- R and probed with anti-P-Tyr antibody; (B) cell lysates
prepared from FA-C isogenic lines were immunoprecipitated with
anti-STAT1 and probed with anti-P-Tyr antibody; (C) the blot used for
panel A was stripped and reprobed with anti-STAT1 monoclonal
antibody; (D) the blot used for panel B was stripped and reprobed with
anti-STAT1 monoclonal antibody; (E) the blot used for panel A was
stripped and reprobed with anti-IFN- R ; (F) the blot used for
panel B was stripped and reprobed with anti-STAT1 .
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|
To confirm the above observations, we conducted coimmunoprecipitation
experiments using anti-STAT1 antibody. As shown in Fig.
4B, on the blot
probed with anti-P-Tyr, two tyrosine-phosphorylated
proteins are
evident in both IFN-

-treated normal (JY) and complemented
(HSC536N/FANCC) cell lines but not in stimulated mutant FA-C
(HSC536N
and HSC536N/neo) cell lines (lanes 2 and 6 versus 4 and 8).
The
upper band proved to be IFN-

R

, as indicated in Fig.
4D (lanes
2 and 6), when we reprobed the membrane with antibody to the receptor
protein. Although a small amount of phosphorylated STAT1 was
precipitated
by anti-STAT1 antibody in FA-C cell lines HSC536N and
HSC536N/neo,
no coprecipitating IFN-

R

receptor protein was
detected (Fig.
4B and D, lanes 4 and 8), indicating reduced association
of STAT1
with the receptor in mutant
cells.
As an important technical note, we emphasize that we were unable to
detect STAT1 in anti-IFN-

R

or IFN-

R

in anti-STAT1
immunoprecipitates from cell lysates prepared with RIPA buffer
or NP-40
lysis buffer (see Materials and Methods). Washing of
the
immunoprecipitate in 1% Triton X-100, 1% NP-40, or 1% digitonin
lysis buffer resulted in the disruption of the STAT1-IFN-

R

complex,
suggesting that interaction between STAT1 and the receptor
protein
may be weak or unstable. Finally, Western blot analysis of the
anti-IFN-

R

and -STAT1 immunoprecipitates demonstrated the
presence
of equivalent IFN-

R

and STAT1 proteins in all four cell
lines
at 5 min of IFN-

incubation compared to unstimulated cells
(Fig.
4E and F), indicating that both the receptor and STAT1 proteins
were constitutively expressed at equivalent levels in all isogenic
cell
lines. Taken together, these results demonstrate an unambiguous
requirement for the FANCC protein in the formation of
IFN-

-induced
STAT1

-IFN-

R
complexes.
IFN-
-induced association of STAT1 with FANCC.
The
coimmunoprecipitation result (Fig. 4) demonstrated that FANCC
is required for recruitment of STAT1 to the activated IFN-
R complex.
Because this suggested that the activation of STAT1 might result from a
direct interaction of the FANCC protein with STAT1, we sought to
identify a binding interaction between FANCC and STAT1. JY cells
were stimulated with IFN-
or left untreated, and WCL were incubated
with glutathione-Sepharose bead-bound GST fusion proteins containing
the normal FANCC (GST-FANCC) or a patientderived mutant
FANCC (GST-L554P). FANCC-interacting cellular proteins were
then isolated from WCL by glutathione-Sepharose affinity precipitation.
STAT1 Western blot analysis of the affinity-precipitated proteins (Fig.
5A, top) showed that STAT1 bound strongly
to the normal FANCC fused to GST (lane 4) but not to GST alone
(lanes 1 and 2). STAT1 bound weakly to the GST fusion protein
containing the L554P mutation (lane 6). Reprobing the blot with
antibodies to FANCC revealed comparable amounts of GST-fused
FANCC and L554P proteins were input in the binding reactions (Fig.
5A, bottom). It is important to note that FANCC-STAT1 interaction
was detected only in lysates prepared from IFN-
-stimulated cells
(lanes 3 and 5 versus 4 and 6), suggesting that the association is
initiated by early activation elements of the IFN-
signaling
pathway.

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FIG. 5.
IFN- -induced association of FANCC and STAT1 in B
lymphocytes. (A) WCL (1 mg of protein) of JY cells stimulated with
IFN- for up to 10 min were incubated with yeast-expressed GST fusion
proteins (10 µg) bound to glutathione-Sepharose beads. The
glutathione-Sepharose affinity precipitates were analyzed by SDS-PAGE
followed by immunoblotting with antibodies to STAT1 (top) and
reprobed with anti-FANCC to verify the input of the GST fusion
proteins. (B) Coimmunoprecipitation (IP) of endogenous FANCC and
STAT1. WCL prepared from JY and HSC536N cells (500 µl of each,
containing 1 mg of total proteins) were incubated with
FANCC-specific antibody in the presence of protein A-agarose. The
immunocomplexes were then analyzed by Western blotting with antibody
specific for P-STAT1 (top) and for total STAT1 (bottom).
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|
Because STAT1 interacted in vitro with GST-FANCC, we sought to
determine whether similar association also occur within cells.
To
detect complexes formed between endogenous FANCC and STAT1
in
IFN-

-stimulated lymphocytes, we performed immunoprecipitation
with
an affinity-purified FANCC-specific antiserum. The
precipitates
were analyzed by Western blotting with antibodies against
either
P-STAT1 or total STAT1. As shown in Fig.
5B, a detectable
P-STAT1
signal was obtained in precipitates from IFN-

-stimulated JY
and
HSC536N/FANCC cells, which was absent in samples from the
stimulated
mutant HSC536N cells (lanes 2 and 6 versus 4). There was no
detectable
P-STAT1 in precipitates from unstimulated cells (lanes 1, 3, and
5). This result was further substantiated by reprobing the blot
with an antibody against total STAT1. The lower panel of Fig.
5B
clearly demonstrated that FANCC coimmunoprecipitated with STAT1
in
the extracts from the normal JY and corrected HSC536N/FANCC
cells treated with IFN-

(Fig.
5B, bottom, lanes 2 and 6). We
also
found that there was significantly reduced total STAT1 bound
to
FANCC in the mutant HSC536N/neo extracts (lane 4), consistent
with
our observation that STAT1 bound weakly to the GST fusion
protein
containing the mutant FANCC L554P (Fig.
5A, lane 6). Faint
bands
were detected in the precipitates from unstimulated cell
extracts when
the blot was reprobed with antibody against total
STAT1 (Fig.
5B,
bottom, lanes 1, 3, and 5). This was probably
due to a slight
reactivity of the antiserum with a protein that
was migrating at the
same position as the STAT1 protein, as we
observed the same faint
band in a preimmune precipitation (data
not shown). To assess
whether the FANCC mutation directly caused
a failure of
STAT1-FANCC binding, we performed a binding assay
using WCL from
HSC536N cells. As shown in Fig.
6B, STAT1 from
IFN-stimulated HSC536N
cells was able to bind to a normal GST-FANCC
with association
kinetics comparable to that observed in the normal
JY cells (Fig.
6A), indicating that the limiting factor
for STAT1
complex formation in IFN-stimulated FA-C mutant cells is
FANCC
itself.

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FIG. 6.
Kinetics of FANCC-STAT1 association. WCL (1 mg of
total proteins) of JY (A) and HSC536N (B) cells stimulated with IFN-
(10 ng/ml) for various periods of time were incubated with
GST-FANCC (10 µg) bound to glutathione-Sepharose beads, and the
glutathione-Sepharose affinity precipitates were analyzed by
immunoblotting with antibodies to P-STAT1 (A and B, top), STAT1 /
(A, middle; B, bottom), and FANCC (A, bottom). Lanes 1 and 2 in
panel A and lanes 1 to 4 in panel B are the input controls, each of
which was loaded with 100 µg of total WCL proteins. The double bands
in lanes 1 and 2 of the bottom gel in panel A are nonspecific. (C) In
vivo phosphorylation of STAT1 in cells stimulated with IFN- . JY and
HSC536N cells were metabolically labeled with
[32P]orthophosphoric acid for 4 h followed by
IFN- treatment for the indicated time points. WCL were prepared and
affinity precipitated with GST-FANCC and analyzed by SDS-PAGE and
autoradiography (top). Western blot analysis, performed on these
affinity precipitates to determine the amounts of
tyrosine-phosphorylated and total STAT1 proteins, is shown in the lower
three panels, which were probed with antibodies specific for P-STAT1,
STAT1, and FANCC, respectively. The order of samples is the same in
all panels.
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|
IFN-
initially induces FANCC association with STAT1 form not
phosphorylated on Y701.
We further examined
kinetically whether the interaction of FANCC-STAT1 in induced cells
depended on prior tyrosine phosphorylation of STAT1. We stimulated JY
cells with IFN-
for various periods of time and performed GST fusion
protein affinity binding assays. FANCC-bound STAT1 could be
detected as early as 30 s after IFN-
treatment, reached its
maximal level at 5 min, and decreased at 30 min after stimulation (Fig.
6A, top). Interestingly, the amounts of total STAT1 proteins associated
with FANCC were comparable between 30 s and 1 min of IFN-
treatment, but the amount of phosphorylated STAT1 bound to FANCC at
the 30-s time point was negligible compared to that at the 1-min time
point (Fig. 6A, top and middle, compare lanes 4 and 5). While we cannot
rule out the possibility that the antibody against total STAT1 may have
higher affinity than the one specific for P-STAT1, comparison of
P-STAT1 and total STAT1 at the 30-s, 1-min, and 5-min time points
clearly demonstrated an increase in the phosphorylated form that occurs
only following full binding of the nonphosphorylated form of STAT1.
Because the kinetic appearance of STAT1 at the docking site occurs at
the time of phosphorylation, our results are most compatible with the
notion that IFN-
initiates the association of FANCC with STAT1
before STAT1 becomes phosphorylated and that the association of
FANCC with P-STAT1 results from the delivery by FANCC to the IFN-
R docking site. Because STAT1 is also phosphorylated on serines (e.g., Ser727), additional studies using
[32P]orthophosphate-labeled cells were conducted to
confirm that the initial association of STAT1 with FANCC is with an
unphosphorylated form. Figure 6C (top) shows that significantly more
P-STAT1 was bound to GST-FANCC in IFN-stimulated JY cells at 20 min
than at the 1-min time point. However, the amounts of FANCC-bound
P-STAT1 and total STAT1 were comparable between these two time points (Fig. 6C, middle, compare lanes 2 and 3). This suggests that the more
intense signal obtained at 20 min of IFN-
stimulation is most likely
due to serine phosphorylation. In HSC536N cells, there was no P-STAT1
bound to GST-FANCC at 1 min of IFN stimulation, but P-STAT1 was
evident at the 20-min time point. Interestingly, however, total bound
STAT1 could be detected in equal amount at both 1- and 20-min time
points even though none of these bound STAT1 proteins were tyrosine
phosphorylated. Taken together, these results suggest that the
association of STAT1 with FANCC precedes STAT1 phosphorylation.
FANCC-STAT1 association in MO7e cells and its functional
relevance.
We next sought to determine whether FANCC-STAT1
association occurs in normal myeloid precursor cells. MO7e cells were
incubated in the presence of IFN-
, and analysis of the WCL by
Western blotting indicated that IFN-
induced tyrosine
phosphorylation of STAT1 in MO7e cells (Fig.
7A, top, lane 2). A GST fusion protein
affinity binding assay showed that IFN-
induced association of STAT1
with the normal FANCC protein but not with the mutant L554P protein fused to GST (Fig. 7A, top and middle, lanes 4 versus 6). Figure 7A
demonstrates that comparable amounts of total STAT1 protein (middle,
lanes 1 and 2) and GST fusion proteins (bottom, lanes 3 to 6)
were loaded in the binding reactions.

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FIG. 7.
FANCC-STAT1 association in stimulated MO7e cells.
(A) WCL of MO7e cells stimulated with IFN- (10 ng/ml) for 10 min
were used for either direct Western blotting (60 µg of total WCL
proteins; lanes 1 and 2) or affinity precipitation (1 mg of total WCL
proteins) with yeast-expressed GST fusion proteins (10 µg) and then
analyzed by immunoblotting (lanes 3 to 6). The blot was probed with
anti-P-STAT1 (top), anti-STAT1 (middle), or anti-FAC (bottom). (B) MO7e
cells were stimulated with IFN- (20,000 U/ml), GM-CSF (100 U/ml), or
SCF (50 ng/ml) for 10 min, and WCL was subjected to either direct
Western analysis (100 µg of total WCL proteins; lanes 1 and 2) or GST
affinity binding (1 mg of total WCL proteins; 10 µg of GST fusion
proteins; lanes 3 to 6) as described above. All IFN- , GM-CSF, and
SCF blots were first probed with anti-P-STAT1 and then stripped and
reprobed with anti-STAT1. (C) MO7e and MO7ER cells were treated with
GM-CSF (100 U/ml) and EPO (40 U/ml), respectively, for 10 min. WCL was
then subjected to either direct Western blotting (100 µg of total WCL
proteins; lanes 1 and 2) or GST affinity binding (1 mg of total WCL
proteins; 10 µg of GST fusion proteins; lanes 3 to 6) as described
for panel A. The GM-CSF blot was probed first with anti-P-Stat5 and
then anti-Stat5; the EPO blot was probed first with anti-P-Stat3 and
then anti-Stat3.
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|
Although the STAT1-FANCC association is clear-cut, and while we can
state with reasonable certainty that the hypersensitivity
does not
require STAT1 activation, the mechanism of IFN hypersensitivity
in FA-C
cells is less clear and seems even counterintuitive. The
STAT1
activation defect in FA cells may play a more direct role
in signaling
for survival or self-replication. Certain growth
and survival factors
for hematopoietic progenitor cells and stem
cells are known to activate
STAT1. These include SCF (
5,
7),
GM-CSF (
37),
G-CSF (
1,
47), and EPO (
24,
36). Our hypothesis
that the STAT1 defect in FA cells might result in a functional
disconnection from survival cues required, first, that FANCC and
STAT1 associate in growth factor-responsive hematopoietic cells
after
stimulation with growth and survival factors other than
IFN-

. To
this end, we stimulated MO7e cells with IFN-

, GM-CSF,
and SCF, all
of which have been shown to induce tyrosine phosphorylation
of STAT1
(
6,
7,
24) and stimulated MO7ER cells with EPO.
As shown in
Fig.
7B, IFN-

, GM-CSF, and SCF induced activation
of STAT1 by
tyrosine phosphorylation (lane 2) and association
of STAT1 and
FANCC proteins (lane 4). The intensity of the signal
owing to STAT1
phosphorylation was greatest for IFN-

and least
for GM-CSF (Fig.
7B,
lane 2, compare between panels). As has been
described by others
(
24), we also noted low-level STAT1 phosphorylation
in MO7ER
cells in response to EPO (data not shown). To extend
the above
observations, we sought to determine whether the FANCC
protein also
interacts with other STAT molecules. MO7e and MO7ER
cells were treated
with GM-CSF and EPO, respectively. Both GM-CSF
and EPO have been
reported to activate STAT3 (
7), and the former
also induces
activation of STAT5 (
34) in these cells. Figure
7C
demonstrates that GM-CSF and EPO activate Stat5 and Stat3,
respectively
(lane 2), but that no FANCC-Stat5 or FANCC-Stat3
association
was detected in these
cells.
Because Steel factor deficiencies account for both bone marrow and germ
cell deficiencies in mice, both of which occur in
FANCC knockout
mice, we sought to test the hypothesis that FANCC
mutations might
result in unresponsiveness to Steel factor, a
survival factor for
hematopoietic cells that induces STAT1 activation
(
5,
7). We
treated BM cells from heterozygotes and FANCC
knockout mice with
graded doses of Steel factor in the presence
of EPO or IL-3 in clonal
assays. As shown in Fig.
8A, both normal
(heterozygote FANCC
/+) and mutant
(FANCC
/
) BM cells proliferated in response to the
hematopoietic growth
factor SCF, but clonal growth of hematopoietic
progenitor cells
from FANCC
/
animals was
consistently less responsive to SCF stimulation,
confirming earlier
preliminary reports (
3). We found the same
to be true in
clonal assays from marrows of STAT1
/
mice (Fig.
8B).
However, clonal growth of progenitor cells from
STAT1
/
mice differed from that from FANCC
/
mice in that
they were completely resistant to IFN-mediated suppression
(Fig.
8C).

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FIG. 8.
SCF induces CFU-GM growth of murine BM cells. (A) BM
cells from FANCC /+ and FANCC /
mice were exposed to either recombinant murine SCF (0 to 10 ng/ml) or a
combination of three growth factors (SIE): murine Steel factor (10 ng/ml), murine IL-3 (10 ng/ml), and human EPO (2 U/ml). CFU-GM and
BFU-E were counted after 14 days. Data points represent mean colony
growth (±SD) of triplicate cultures. (B) Suboptimal responses of
erythroid burst-forming units (BFU-E) from STAT1 / to
EPO and SCF. Results of a single representative experiment (or two
independent experiments) are shown. Data points represent mean (±SD)
clonal growth derived from colony counts in three replicate plates. Control BFU-E growth was
defined in cultures containing IL-3 (10 ng/ml), Steel factor (10 ng/ml), and EPO (2 U/ml). Control BFU-E growth was equivalent in
heterozygotes (31 colonies 105 cells) and
STAT1 / mice (48 colonies/105 cells). (C)
Suppression of BFU-E growth by IFN- is STAT1 dependent. Optimally
stimulated (IL-3, SCF, and EPO) clonal cultures of erythroid marrow
cells from STAT1 / mice and control animals were
exposed to the indicated doses of IFN- throughout the
culture period. Results shown are from a single representative
experiment. Each point represents mean (±SD) BFU-E growth
expressed as percent control. Control colony growth was
equivalent (see above), but IFN- failed to suppress clonal growth in
STAT1 / mice, even at supraphysiological levels.
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 |
DISCUSSION |
We have recently demonstrated that hematopoietic progenitor cells
from mice nullizygous at the FANCC locus and children with FA-C are
hypersensitive to the antiproliferative effect of IFN-
(38,
55). Because most of the known effects IFN-
are ignited via
STAT1 activation and nuclear translocation of STAT1 homodimers, we
initially expected that at IFN-
doses too low to activate STAT1 in
normal cells, FA-C cells would contain high levels of phosphorylated
STAT1. We were surprised to discover that in FA-C cells, STAT1
phosphorylation in response to IFN-
was reduced markedly (10,
38). We ruled out the possibility that reduced STAT1 activation
per se was the cause of progenitor cell hypersensitivity to IFN-
by
quantifying growth of clonogenic progenitor cells from the marrow of
STAT1
/
mice. Progenitors from these mice were
completely resistant to the growth-suppressive activity of IFN-
(Fig. 8C), indicating that the hypersensitivity derived not from the
failure of STAT1 activation but from another biological effect of
FANCC mutation. Like FA-C progenitors, however, progenitor cells
from STAT1
/
mice were suboptimally responsive to
combinations of Steel factor (SCF) and EPO (Fig. 8B), indicating that
inactivating FANCC mutations may blunt responses to survival
factors for hematopoietic progenitor cells.
We therefore sought to more fully understand the function of the
FANCC protein vis-à-vis the JAK/STAT signaling pathway, using
the IFN-
signaling pathway as a paradigm. We also investigated factor-dependent hematopoietic cell lines, seeking to reveal
FANCC-JAK-STAT relationships by using other receptor molecules
including SCF, EPO, and GM-CSF. We reasoned that the normal FANCC
protein, localized primarily in the cytoplasm (16, 56, 57),
might functionally interact with key components of the JAK/STAT
cytokine signaling cascades induced by survival factors for
hematopoietic cells, and that mutations of FANCC might interdict
such responses. We have confirmed that notion in this study. We used
isogenic cells derived from patients and from nullizygous mice
carrying inactivating mutations in the FANCC gene to dissect the
ligand-induced assembly of the multimeric IFN-
R-JAK-STAT complex.
We confirmed that STAT1 tyrosine phosphorylation was deficient in FA-C
cells (Fig. 1) but determined that the activation kinetics of
IFN-
R
(Fig. 2) and JAK1 and JAK2 (Fig. 3) were normal in FA-C
cells. In addition, immunoblot analysis of total cell lysates prepared
from unstimulated and IFN-
-treated cells indicated that both normal
and FA-C cells constitutively expressed equivalent levels of
IFN-
R
, JAK1, and JAK2. These results suggest that the deficiency
in STAT1
tyrosine phosphorylation results neither from reduced
expression nor activation of the JAK molecules or IFN-
R
. However,
STAT1
was detected only in the IFN-
R complexes of cell lysates
from IFN-stimulated normal and corrected FA-C cell lines (Fig. 4).
Moreover, GST-fused FANCC was shown to bind STAT1 contained in
IFN-
-induced cell lysates, but STAT1 did not bind to a FANCC
mutant protein (a point mutant known to inactivate the function of
FANCC) fused to GST (Fig. 5). Using activation state-specific
antibodies and orthophosphate labeling studies, we have concluded
that FANCC-STAT1 association did not require STAT1
phosphorylation, on either tyrosine or serine residues (Fig. 6). We
further demonstrated that GST-FANCC-STAT1 association was also
induced by IFN-
, IFN-
, GM-CSF, and SCF in MO7e cells and that
mutant GST-FANCC-L554P did not associate with STAT1 in cells stimulated with these factor (Fig. 7). That survival of committed progenitor cells exposed to graded doses of SCF was suppressed in FA-C
progenitor cells from FANCC knockout mice confirmed that FANCC
augments responses to SCF and EPO in hematopoietic cells (Fig. 8).
Our observation that STAT1
forms a complex with IFN-
R
in
normal cells stimulated with IFN-
is consistent with the previous reports that STAT1
physically associates with IFN-
R
both in vitro and in vivo (12, 13, 42). In these reports, STAT1 was
shown to specifically recognize a phosphorylated tyrosine residue in
the cytoplasmic domain of the receptor protein and to
coimmunoprecipitate with IFN-
R
in stimulated cell lysates. Once
recruited into the dimeric IFN-
R
/
complex, STAT1 becomes activated by tyrosine phosphorylation (4, 50, 52). We found that coimmunoprecipitation of STAT1 with IFN-
R
was ligand
dependent and additionally depended on the presence of an intact
FANCC protein (Fig. 4). The difference in STAT1-IFN-
R
coimmunoprecipitation between normal and FA-C cells (Fig. 4A and B) was
not due to the varying levels of the STAT1 proteins, since STAT1 was
expressed at comparable levels (Fig. 1B and 4F). Therefore, it appeared that the presence of a functional FANCC protein somehow facilitated the recruitment of STAT1 to the activated IFN-
R complex.
The reason for the complete failure of STAT1 to appear at the docking
site of the IFN-
R and subsequently to be activated by tyrosine
phosphorylation in FA-C cells is not completely understood. Three
possible mechanisms may account for our observation. First, because
FANCC and STAT1 associate very rapidly and prior to STAT1 phosphorylation (Fig. 6), FANCC itself might function as a
chaperone for STAT1 molecules, moving them from cytoplasm to the
receptor docking sites. This shuttling function of FANCC might
occur directly or with the help of intermediary molecules. Second, that
FANCC interacts with transcription factors including STAT1 in the
studies described here and PLZF homologs (17) is compatible
with the notion that in FA cells genes whose products govern STAT1
docking and activation might be derepressed. Third, FANCC may
suppress the function of proteins that function to inhibit STAT1
activation. There is growing evidence that FANCC interacts with
other FA proteins to form a functional nuclear complex (11,
53). It is possible that such nuclear complex functions to
modulate gene expression in response to external signals such as IFNs
or other cytokines and growth factors. SOCS1, a STAT1-responsive gene
whose product interdicts IFN-induced activation of STAT1, is an
appealing candidate, particularly since SOCS1 nullizygous mice like
FANCC nullizygous mice exhibit IFN-
hypersensitivity (15,
29). However, this molecule functions by reducing JAK activation
(41), and the studies described herein demonstrate that
FANCC mutations result in STAT1 inactivation without reduction in
JAK1 and JAK2 phosphorylation. Consequently, derepression of SOCS1
expression per se is an unlikely explanation for our results.
Nonetheless, while their function is unclear to date, a number of
proteins with C-terminal SOCS domains have been identified
(15), and it remains quite possible that such proteins or
others like them function to inhibit STAT1 activation and are also
encoded by genes responsive to transcriptional repressors with which
FANCC interacts.
Because the initial association of FANCC and STAT1 did not require
tyrosine phosphorylation, we sought to determine whether the initially
bound STAT molecule was phosphorylated on other residues. We found that
FANCC-STAT1 association required neither tyrosine nor serine
phosphorylation (Fig. 6A and C). We also found that STAT1 from induced
FA-C cells was capable of binding to normal GST-FANCC (Fig. 6B),
demonstrating that early IFN-induced signals influence the receptivity
of STAT1 to FANCC, even in mutant cells. Thus, although the nature
of the early IFN-induced signaling pathway is unclear at this time, the
limiting factor for STAT1 phosphorylation in FA-C cells is clearly
FANCC itself, not a FANCC-dependent cofactor. The association
of FANCC with STAT1 prior to receptor docking is one of the
earliest reported responses to IFN-
.
While IFN-
was used as our paradigm, we were also able to
demonstrate the association of STAT1 with FANCC in cells exposed to
hematopoietic growth and survival factors (Fig. 7A and B). Our results
also demonstrate reasonable specificity of FANCC-STAT1 interaction
and potential functional consequence of this interaction. While the
FANCC-STAT1 binding phenomenon occurred in both myeloid and
lymphoid cells (Fig. 7A and B) and occurred in cells stimulated with
IFN-
, IFN-
, GM-CSF, and SCF (Fig. 7A and B), FANCC did not
associate with activated STAT3 and STAT5 (Fig. 7C). Therefore, these
results suggest that although other STAT molecules are present and
activated in appropriately stimulated cells, STAT1 is a specific FANCC partner.
Others have reported that SCF-stimulated BM colony growth is defective
in FA patients (3). Since we have shown that SCF induces
FANCC-STAT1 interaction in MO7e cells, our results are consistent with a role of the interaction in cell proliferation induced by SCF and certain other hematopoietic growth factors. This
notion is further strengthened by our findings that BM progenitor cells
from FANCC
/
and STAT1
/
mice
exhibited blunted responses to SCF. This suggests a potential role of
the FANCC-STAT1 interaction in stem cell and progenitor cell
survival mediated by SCF and possibly other growth factors.
That constitutive activation of IFN-responsive genes like IRF-1 and MxA
(9, 27) is disconnected with STAT1 activation in FA-C cells
not only indicates that the FANCC protein is multifunctional in its
capacity to suppress hematopoiesis but also explains why FA patients
are not susceptible to opportunistic infections as might be predicted
by extrapolation from observations of STAT1
/
mice
(9, 28). That is, some of the constitutively expressed proteins in FA mediate the antiviral state (27, 37, 46) and
would likely be protective. While the mechanisms by which FANCC
mutations result in constitutive activation of ordinarily repressed genes are unclear, that FANCC binds to a PLZF homolog (17) suggests that the failure of FAZF-FANCC interaction
in mutant cells may result in selective gene derepression.
From the hematopoietic standpoint, FA-C cells are hypersensitive to a
variety of mitogenic inhibitory molecules and apoptotic cues, including
cytokines that utilize pathways not known to utilize STAT1
(13). Consequently, we do not argue that the function of FANCC vis-à-vis STAT1 is sufficient to account for
every element of the FA phenotype. If this were the case, we would
need to propose that STAT1
/
mice should share this
phenotype, and they do not. It is clear, however, that FA-C cells,
whose receptor complexes are disconnected from STAT1 signaling
pathways, are likely deprived of survival signals that utilize this
pathway. With STAT1 deficiency alone, this state of disconnection from
survival cues may not be sufficient to cause BM failure (a universal
finding in FA), but when combined with the constitutive expression of
mitogenic inhibitory and apoptotic factors, and hypersensitivity to IFN
and tumor necrosis factor (38), the impact of STAT1
disconnection in hematopoietic cells may be substantial. Additional
studies are warranted on the structural elements of FANCC required
for STAT1 activation and both IRF-1 and MxA repression. We also argue
that a comprehensive investigation of molecular hematopoietic control
is warranted in STAT1
/
mice, particularly using factors
known to influence expansion, survival, and responsiveness of myeloid
and erythroid progenitor cells.
 |
ACKNOWLEDGMENTS |
We thank Manuel Buchwald for providing the lymphoblast cell line
HSC536N from a type C Fanconi anemia patient, A. D. Miller for the
retroviral vector pLXSN, David C. Hinkle for the pGST vector, Brian
Druker for antiphosphotyrosine antibody 4G10, and Tara Koretsky for
valuable technical assistance. We also thank Markus Grompe for the
FANCC mutant cell lines, knockout mice, and helpful discussions.
This work was supported by NIH grant HL48546 and a Department of
Veterans Affairs merit review grant to G.C.B.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Oregon Cancer
Center, Oregon Health Sciences University, Portland, OR 97201. Phone: (503) 494-6343. Fax: (503) 494-7086. E-mail:
grover{at}ohsu.edu.
 |
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Molecular and Cellular Biology, July 2000, p. 4724-4735, Vol. 20, No. 13
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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(2003). Increased sensitivity of Fancc-deficient hematopoietic cells to nitric oxide and evidence that this species mediates growth inhibition by cytokines. Blood
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Tischkowitz, M D, Hodgson, S V
(2003). Fanconi anaemia. J. Med. Genet.
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Pang, Q., Christianson, T. A., Keeble, W., Koretsky, T., Bagby, G. C.
(2002). The Anti-apoptotic Function of Hsp70 in the Interferon-inducible Double-stranded RNA-dependent Protein Kinase-mediated Death Signaling Pathway Requires the Fanconi Anemia Protein, FANCC. J. Biol. Chem.
277: 49638-49643
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Bogliolo, M., Cabre, O., Callen, E., Castillo, V., Creus, A., Marcos, R., Surralles, J.
(2002). The Fanconi anaemia genome stability and tumour suppressor network. Mutagenesis
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Rio, P., Segovia, J. C., Hanenberg, H., Casado, J. A., Martinez, J., Gottsche, K., Cheng, N. C., Van de Vrugt, H. J., Arwert, F., Joenje, H., Bueren, J. A.
(2002). In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice. Blood
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Haq, R., Halupa, A., Beattie, B. K., Mason, J. M., Zanke, B. W., Barber, D. L.
(2002). Regulation of Erythropoietin-induced STAT Serine Phosphorylation by Distinct Mitogen-activated Protein Kinases. J. Biol. Chem.
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Rutherford, T. R., Myatt, N. E., Gibson, F. M., Clarke ;, A. A., Bagby, G. C. Jr
(2002). The Fanconi anemia cell line HSC536N is not sensitive to interferon-gamma and does not cleave PARP in response to Fas-mediated cell killing. Blood
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Kirito, K., Nakajima, K., Watanabe, T., Uchida, M., Tanaka, M., Ozawa, K., Komatsu, N.
(2002). Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation. Blood
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Yagasaki, H., Adachi, D., Oda, T., Garcia-Higuera, I., Tetteh, N., D'Andrea, A. D., Futaki, M., Asano, S., Yamashita, T.
(2001). A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA. Blood
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Pang, Q., Christianson, T. A., Keeble, W., Diaz, J., Faulkner, G. R., Reifsteck, C., Olson, S., Bagby, G. C.
(2001). The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality. Blood
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Hadjur, S., Ung, K., Wadsworth, L., Dimmick, J., Rajcan-Separovic, E., Scott, R. W., Buchwald, M., Jirik, F. R.
(2001). Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase. Blood
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Fagerlie, S. R., Diaz, J., Christianson, T. A., McCartan, K., Keeble, W., Faulkner, G. R., Bagby, G. C.
(2001). Functional correction of FA-C cells with FANCC suppresses the expression of interferon {gamma}-inducible genes. Blood
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Pang, Q., Keeble, W., Diaz, J., Christianson, T. A., Fagerlie, S., Rathbun, K., Faulkner, G. R., O'Dwyer, M., Bagby, G. C. Jr
(2001). Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon {gamma}, tumor necrosis factor-{alpha}, and double-stranded RNA. Blood
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Rathbun, R. K., Christianson, T. A., Faulkner, G. R., Jones, G., Keeble, W., O'Dwyer, M., Bagby, G. C.
(2000). Interferon-gamma -induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members. Blood
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