From the Division of Molecular and Cellular
Immunology, Medical Institute of Bioregulation, Kyushu University,
3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan and the
§ Department of Immunology, Graduate School of
Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Received for publication, November 4, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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As a c-fms-interacting protein, we
cloned a novel adaptor molecule, signal-transducing adaptor protein-2
(STAP-2), which contains pleckstrin homology- and Src homology
2-like (PH and SRC) domains and a proline-rich region. STAP-2 is
structurally related to STAP-1/BRDG1 (BCR downstream signaling-1),
which we had cloned previously from hematopoietic stem cells. STAP-2 is
a murine homologue of a recently identified adaptor molecule, BKS, a
substrate of BRK tyrosine kinase. STAP-2 was tyrosine-phosphorylated
and translocated to the plasma membrane in response to epidermal growth
factor when overexpressed in fibroblastic cells. To define the
function of STAP-2, we generated mice lacking the STAP-2 gene.
STAP-2 mRNA was strongly induced in the liver in response to
lipopolysaccharide and in isolated hepatocytes in response to
interleukin-6. In the STAP-2 Tyrosine kinases play an important role in regulating cell growth,
differentiation, and transformation. Activated receptor tyrosine
kinases trans-phosphorylate several tyrosines in their cytoplasmic
domains, which provide recognition sites for various adaptor and
effector proteins in multiple signal transduction pathways (1, 2).
These adaptor proteins utilize their Src homology-2
(SH2)1 and SH3 domains to
mediate the interactions that link different proteins involved in
signal transduction. For example, the adaptor protein Grb2 links a
variety of surface receptors to the Ras/MAP kinase signaling cascade.
Grb2 interacts with activated receptor tyrosine kinases via its SH2
domain and recruits the guanine nucleotide-releasing factor, SOS
(Son of Sevenless), close to its target protein, Ras, at the cell
membrane. Phosphoinositide-3-OH kinase (PI3K) and phospholipase C Signal transducer and activator of transcription (STAT) family proteins
were identified in the last decade as transcription factors that are
critical in mediating virtually all cytokine signaling (3, 4). These
proteins become activated through tyrosine phosphorylation, which
typically occurs through cytokine receptor-associated kinases, the
Janus kinase (JAK) family proteins (5). However, many reports suggest
that STATs are activated by receptors unrelated to cytokines. For
example, angiotensin II has been shown to activate the JAK/STAT pathway
by unknown mechanisms (6), and we recently reported that STAT3 is
activated by the hepatitis type C virus core protein (7). Usually,
STAT3 is activated by IL-6-related cytokines, IL-10 and the granulocyte colony-stimulating factor (4). Moreover, STAT 3 was also activated by
many tyrosine kinases unrelated to those cytokines, including v-Src,
the EGF receptor, and c-Kit (8, 9). Cytokine receptors that activate
STAT3 possess a YXXQ motif in the cytoplasmic region, which
recruits STAT3 to the receptor (10). Most tyrosine kinases that
activate STAT3 do not possess this motif. A mutation in the sequence
Y933VPL, present in c-Eyk, which is a member of the
Axl/Tyro3 subfamily, to the v-Eyk sequence Y933VPQ led to
increased activation of STAT3 and increased transformation efficiency
(11). Therefore, an adaptor protein that recruits STAT3 to the receptor
tyrosine kinases or Src-like tyrosine kinases may be required for
efficient STAT3 activation. However, so far, no such adaptor molecule
that can recruit STAT3 has been identified.
In the present study, we cloned an adaptor molecule, STAP-2, by yeast
two-hybrid screening of a fetal liver cDNA library using c-fms as bait. STAP-2 contains a PH domain, an SH2-like
domain, and a C-terminal proline-rich region. STAP-2 is a murine
homologue of the recently identified adaptor molecule, BKS, a substrate of the Src-type nonreceptor tyrosine kinase, BRK (12). However, the
physiological function of STAP-2/BKS has not been investigated. Therefore, we generated STAP-2/BKS gene-disrupted mice. We also noticed
that STAP-2 contains a YXXQ motif in the C-terminal region and that this motif is conserved among mammals. Therefore, we examined
the role of STAP-2 in the liver at the acute-phase (AP) response, which
is shown to be dependent on the IL-6/STAT3 pathway. We found that
STAP-2 was induced by IL-6 in wild-type primary hepatocytes and that
STAT3 activation as well as the induction of AP response
genes was reduced in the liver of the mutant mice. Using cultured cells
and transient expression systems, we confirmed that STAP-2 potentiated
STAT3 activation through its conserved YXXQ motif. These
data indicate that STAP-2 is a unique signal-transducing adaptor
molecule that may link several tyrosine kinases and STAT3.
Yeast Two-hybrid Screening--
The cytoplasmic domain of
c-fms (codon 560-926) was inserted in-frame into the GAL4
DNA-binding domain vector pBTM116 (13). The fetal liver yeast
two-hybrid cDNA library in pACT2 was purchased from
Clontech. More than 2 × 106
transformants were screened according to the procedure described previously (14).
Generation of the Targeting Vector and the Targeted Embryonic
Stem Cells and Mice--
A genomic library from the 129/SV mouse
strain (Stratagene) was screened with a cDNA probe of the mouse
STAP-2, and several overlapping positive clones, including all 13 exons, were identified. The targeting vector was constructed by
replacing the 4th through 13th exons with a pgk-neo cassette
while preserving 3.8-kb (left arm) and 10.5-kb (right arm) flanks of
homologous sequences (see Fig.4A). The hsv-tk
gene was inserted for negative selection. Homologous recombination in
murine embryonic stem cells was performed as described previously (15)
and confirmed by Southern blot analysis. The chimeric mice were
backcrossed to C57BL/6 five times. The resultant F5 mice were
intercrossed to obtain the offspring for analysis.
Animals and Treatment--
Mice were bred and maintained under
specific pathogen-free conditions. Ten-week-old mice were injected
intraperitoneally once with 2 mg/mouse of lipopolysaccharide (LPS;
Sigma). After the indicated periods, blood samples were collected, and
the mice were sacrificed. The livers were immediately removed and used for total RNA or protein.
Northern Blot and RT-PCR Analysis--
Total RNAs from mouse
liver, primary hepatocytes, primary macrophages, or Hep3B cells were
prepared using the TRIZOL reagent (Invitrogen). RNAs were
separated on 1% agarose, 2.4% formaldehyde gels and then transferred
to Hybond-N+ nylon membranes (Amersham Biosciences). The 0.2-0.4-kb
cDNA probes of murine STAP-2, serum amyloid P component (SAP),
haptoglobin, and GAPDH were prepared using polymerase chain reaction
with reverse-transcribed liver first-strand cDNAs as the template.
STAP-2 primers were used as indicated below (for RT-PCR). The filters
were preincubated for 1 h at 65 °C and incubated overnight at
65 °C with a radiolabeled probe in a hybridization solution (80 mM Tris-HCl, pH 8, 4 mM EGTA, 0.6 M
NaCl, 0.1% SDS, 10× Denhardt's solution, 100 µg/ml salmon sperm
DNA). The filters were washed three times with 0.1× SSC and 0.1% SDS
at 65 °C and analyzed by autoradiography. For RT-PCR, first-strand
cDNAs were prepared using Superscript II reverse transcriptase
(Invitrogen) following the manufacturer's instructions. The specific
primer set for mouse STAP-2 cDNA used was
5'-TGAGGCTCTGCTGGGAAGCTCACG-3'/5'-GGGAGACCCATTGAGAATCTGCCG-3'.
Immunoprecipitation and Immunoblotting--
Cells were grown in
6- or 10-cm dishes and lysed in 1 ml of radioimmune precipitation
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented
with phosphatase and protease inhibitors. For the binding assays,
immunoprecipitation or GST-IBD was performed. Cell lysates were
incubated for 1.5 h at 4 °C with 20 µl (50% v/v) of protein
A-Sepharose (Amersham Biosciences) with 1 µg of rabbit anti-STAT3
(C-20) antibody (Santa Cruz Biotechnology) or 20 µl (50% v/v) of
GSH-Sepharose beads (Amersham Biosciences) and then washed six times
with a radioimmune precipitation buffer. For the tyrosine
phosphorylation assay, v-Src or GST-JH1 cDNAs were co-transfected
to HEK-293 cells with each YF mutant of STAP-2. After 24 h, cells
were lysed and immunoprecipitated with 1 µg of anti-Flag M2 antibody
as described above. For immunoblotting, samples were separated with
10% SDS polyacrylamide gel. Proteins were transferred to Hybond ECL
nitrocellulose membranes (Amersham Biosciences) and then probed with
anti-Flag M2, anti-STAT3 (Santa Cruz Biotechnology),
anti-phospho-Tyr-705-STAT3 (Cell Signaling Technology), or
anti-phosphotyrosine (4G10) antibodies as described (16). Total
proteins from mouse liver were prepared as described previously
(17).
Recombinant Plasmid and Proteins--
cDNAs of human STAP-2
and mutants were generated using polymerase chain reaction (PCR) with
wild-type human STAP-2 cDNA as the template and then cloned into
the mammalian expression vector pcDNA3 (Invitrogen). Four tyrosine
mutants were prepared using PCR-based point mutation. Deletion mutants
lacking the PH domain ( Cell Culture and Transient Transfection--
HEK-293 cells, A431
cells, and Hep3B cells were maintained in Dulbecco's modified Eagle's
medium with 10% fetal calf serum. MCF7 cells were kindly provided by
Dr. T. Furukawa (Kagoshima University) and maintained in RPMI 1640 containing 10% fetal calf serum, 0.1 mM nonessential amino
acids, and 0.01 mg/ml bovine insulin. For the luciferase assay, the
acute-phase response element (APRE) and Elk-1 reporter plasmids
(Promega) (16, 18) and several STAP-2 constructs with v-Src or
EGF receptor cDNAs were transfected using a Fugene6
transfection reagent (Roche Diagnostics) following the manufacturer's
instructions. A Isolation and Culture of Hepatocytes and
Macrophages--
Primary hepatocytes were prepared using the two-step
collagenase perfusion method as described (19). After isolation, the cells were washed two times in Williams' E medium with 10% fetal bovine serum, L-glutamine (Sigma), 100 nM
dexamethasone (Sigma), and 1 µM insulin (Sigma). The
cells were centrifuged at 600 rpm for 1 min between washes. Cell
viability, as estimated by trypan blue exclusion, was routinely more
than 90% following this procedure. The cells were plated on collagen
type I-coated plates in the above described medium at 3 × 106cells/100-mm dish and incubated in 5% CO2
at 37 °C for 36 h prior to the experiments. Intraperitoneal
macrophages were prepared and cultured as described previously (20).
For each experiment, cells were stimulated with LPS (10 ng/ml) (Sigma),
IL-6 (50 ng/ml, Calbiochem), or IL-1 Measurement of Nucleotide Accession Number--
The nucleotide sequence
reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI
nucleotide sequence data bases under accession number
AW049765.
Sequence Comparison of STAP/BRDG/BKS
Family Proteins--
Using a yeast two-hybrid screening with
an oncogenic c-fms kinase domain as bait, we isolated a
cDNA clone, designated STAP-2, from a fetal liver library.
Full-length human and mouse cDNA of STAP-2 was isolated by cDNA
library screening by RACE-PCR. STAP-2 is a murine homologue of BKS, an
adaptor molecule recently identified as a substrate of BRK (12). The
mouse STAP-2 gene encodes a protein of 411 amino acids that is
structurally related to STAP-1 (stem cell-specific
adaptor protein-1) (14). STAP-1 is identical to
the Tec-interacting molecule, BRDG1, which is implicated in a link
between a B-cell receptor and c-fos induction (22). Because STAP-2 is expressed in a variety of tissues, we changed the definition of STAP to signal-transducing
adaptor protein. For a comparison with
STAP-1/BRDG1, we have used STAP nomenclature throughout this paper.
Both STAP-1 and STAP-2 contain an N-terminal PH domain and a region
distantly related to the SH2 domain (overall 33% amino acid identity).
However, STAP-2 has a C-terminal proline-rich region that is not
present in STAP-1. The N-terminal PH domain of STAP-1 and STAP-2 shared
36% identity and 58% similarity. The central region of STAP-2 is
distantly related to the SH2 domain. This region of STAP-2 shares 40%
sequence identity with that of STAP-1 and 29% sequence identity with
the SH2 domain of human PLC
The overall structure of STAP-2 resembles that of docking proteins such
as IRS, GAB, and Dok, because these docking molecules contain a PH
domain at the N terminus, a phosphotyrosine-binding (PTB) domain or a
Met-binding domain (MBD) in the middle, and tyrosine phosphorylation
sites in the C-terminal region.
Tyrosine Phosphorylation and Membrane Localization of the STAP-2
Protein--
From its docking/adaptor protein-like structure, we
expected STAP-2 to be a substrate of tyrosine kinases. To examine this possibility, STAP-2 was co-transfected with constitutively active tyrosine kinases in HEK-293 cells. STAP-2 was strongly phosphorylated by various tyrosine kinases, including v-Src (Fig.
2A-a), a JAK2 tyrosine kinase
domain fused to GST (GST-JH1) (Fig. 2A-b), and an active
c-Kit (D816V) (data not shown).
To determine the phosphorylation site of STAP-2, four predicted
tyrosine residues were mutated to phenylalanine residues. These four YF
mutants (Y22F, Y250F, Y310F, and Y322F) of human STAP-2 were
co-transfected with v-Src or GST-JH1, and the phosphotyrosine state was
investigated by anti-phosphotyrosine (4G10) immunoblotting. The
intensity of the bands was then quantified using a densitometer (Fig.
2A, a and b). Compared with the phosphorylation
levels of wild-type (WT) STAP-2, the Y250F mutant was rarely
phosphorylated by v-Src and GST-JH1, which suggests that the
Tyr-250 residue is important for the interaction with kinases.
The phosphorylation levels of three other tyrosine residues were
different between v-Src and GST-JH1. Y22F and Y322F mutants were much
less phosphorylated by v-Src, and the Y310F mutant was phosphorylated
normally, which suggests that Tyr-22 and Tyr-322 are the major tyrosine
phosphorylation sites by v-Src. On the other hand, the phosphorylation
levels of Y22F, Y310F, and Y322F by GST-JH1 were reduced to 80-60% of the levels of wild-type STAP-2, which suggests that these three are
potential phosphorylation sites by activated JAK2.
Next, we examined the phosphorylation of STAP-2 by natural ligands that
activate tyrosine kinases. We detected the mRNA expression of
endogenous STAP-2 in M1 myeloid cells treated with LIF and NIH-3T3
fibroblastic cells (Fig. 2B). However, because anti-murine STAP-2 antibody was not available, we could not detect the
phosphorylation of endogenous STAP-2 protein. Thus, we generated stable
transformants expressing wild-type STAP-2. In the M1 cells, the STAP-2
protein was phosphorylated after long-term LIF treatment (Fig.
2C-a). We also found that STAP-2 was rapidly (10 min)
tyrosine-phosphorylated in response to EGF in NIH-3T3 expressing
exogenous STAP-2 and the EGF receptor (Fig. 2C-b).
To determine the subcellular localization of STAP-2, we expressed
STAP-2 fused to GFP at the C terminus in A431 cells. As shown in Fig.
3A, GFP-wild-type STAP-2 was
present throughout the cytoplasm and nucleus before stimulation, but
the STAP-2 protein was rapidly (5-10 min) translocated to the plasma
membrane in response to EGF stimulation (Fig. 3A). The
mutant STAP-2 lacking an N-terminal PH domain ( Regulation of Expression of STAP-2 mRNA in the
Liver--
Although STAP-2 has been shown to be relatively widely
expressed in murine and human tissues (data not shown and Ref. 12), the
regulation of STAP-2 expression has not been clarified. Thus, we
examined the 5' region of the STAP-2 genomic sequence by using a data
base (www.motif.genome.ad.jp). We found that the mouse STAP-2
genome contains several potential binding sites for c-Rel, AP-1,
p65/NF- Acute-phase Response of the STAP-2 Knockout Mice--
To further
assess the physiological function of STAP-2 in AP response, we
developed mice with a targeted disruption in the STAP-2 gene locus. To
obtain the loss-of-function mutant, 9 (4th-13th) of 13 exons
containing the last half of the PH domain as well as the entire SH2
domain and the C-terminal region of STAP-2 were deleted (Fig.
5A). The disruption of STAP-2
gene and expression were confirmed by Southern blot analysis (Fig.
5B) and Northern blot analysis of total RNA from the heart
(Fig. 5C), respectively. Offspring were born within the
Mendelian expectation ratio from intercrosses of heterozygotes as well
as incrosses of homozygotes. This indicates that STAP-2 is not
necessary for fertility and development. Adult STAP-2
We then investigated the effect of STAP-2 deficiency on AP response in
the liver. STAP-2+/+ and STAP-2
We then examined AP gene induction in primary hepatocytes isolated from
wild-type or STAP-2-deficient mice. We focused on IL-6, because IL-6
has been shown to play a major role in AP gene induction (35), inducing
STAP-2 mRNA expression by IL-6 in hepatocytes (see Fig.
4B). As shown in Fig. 6B, IL-6-induced SAP
mRNA levels were similar at 1.5 h after stimulation between
STAP-2+/+ and STAP-2
It has been shown that STAT3 plays an essential role in AP gene
induction in the liver (17). To elucidate the mechanisms of STAP-2 on
AP gene induction, we investigated STAT3 activity in the liver after
LPS challenge in vivo or in the primary hepatocytes stimulated with IL-6. As shown in Fig. 6C-a, at 6 h
after LPS challenge, the phosphotyrosine levels of STAT3 were lower
(about 50%) in the mutant mice than in their wild-type littermates.
STAT3 phosphorylation was detected even at 24 h after LPS
challenge in wild-type mice liver, but it was not detectable in
STAP-2-deficient mice liver. In the wild-type primary hepatocytes, the
time course of IL-6-induced STAT3 phosphorylation was biphasic; it
peaked after 30 min-1 h and decreased but was up-regulated again at
6 h (Fig. 6C-b). In the mutant hepatocytes, the
re-up-regulation after 6 h was not observed (Fig.
6C-b). These data suggest that in IL-6-treated hepatocytes
as well as in the liver after LPS challenge, the STAP-2 gene is
involved in the induction of AP genes through STAT3, especially at the
later phase of the responses.
STAP-2 Potentiates Activation of STAT3 in Cultured Cells--
To
confirm the positive regulatory role of STAP-2 on STAT3 activation, we
examined the effect of forced expression of STAP-2 and its mutants on
STAT3 activation. First, we tried the STAT3-dependent APRE-reporter assay in HEK-293 and MCF7 cells (10, 24). Wild-type STAP-2 enhanced EGF- or LIF-induced APRE-reporter activation in HEK-293
cells (Fig. 7A). With the same
conditions, the Elk-1 reporter gene, which reflects MAP kinase
activation (18, 25), was not enhanced by STAP-2 (Fig. 7B).
As shown in Fig. 7C, v-Src-induced APRE-reporter gene
activation was also enhanced by wild-type STAP-2 but not by
STAP-2Y250F and STAP-2Y322F mutants and a
deletion mutant lacking the PH domain (
We next examined the effect of STAP-2 overexpression on IL-6-induced AP
protein in a hepatoma cell line (Fig. 7D). We measured
Because Tyr-322 of human STAP-2 is the potential STAT3-binding site, we
examined the interaction between STAP-2 and STAT3. Flag-tagged
wild-type and mutant STAP-2 were expressed in HEK-293 cells in the
presence of v-Src, and STAP-2 protein co-immunoprecipitated with STAT3
was examined by immunoblotting with an anti-Flag antibody. As shown in
Fig. 7E, wild-type STAP-2 (WT), but not
STAP-2Y250F or STAP-2Y322F mutants, was
associated with STAT3. None of the tyrosine residues of the Y250F
mutant was phosphorylated by v-Src or GST-JH1 (see Fig. 2). Thus, these
data indicate that phosphorylated Tyr-322 is necessary for the
interaction between STAT3 and STAP-2.
In this report, we have characterized a novel adaptor/docking
protein called STAP-2. STAP-1 was previously cloned as a
c-Kit-interacting protein expressed predominantly in
undifferentiated hematopoietic stem cells or myeloid cell lines (14).
STAP-1 also was identified as a Tec-interacting protein that is
tyrosine-phosphorylated in response to B-cell receptor stimulation,
termed BRDG1 (22). BRDG1 was shown to participate in a positive
feedback loop by increasing the activity of Tec. In this study, we
found that STAP-2, rather than MAP kinase, is involved in the
activation of STAT3. STAP-2 contains a YXXQ motif, and its
mRNA was induced by IL-6 in primary hepatocytes. Furthermore, we
showed that late-phase activation of STAT3 as well as AP gene induction
was impaired in STAP-2 The overall structure of STAP-2 resembles that of docking proteins such
as IRS, GAB, and Dok. All docking proteins contain the N-terminal
membrane-targeting domain and a large region that contains multiple
binding sites for the SH2 and SH3 proteins in the C-terminal region
(26-28). Some docking proteins are associated with the cell membrane
by a myristyl anchor (e.g. FRS2) or a transmembrane domain
(e.g. LAT) (29). However, most docking proteins contain a PH
domain at their N terminus that interacts with phosphatidylinositol 1,4,5-trisphosphate in response to agonist-induced stimulation of PI3K (30). Translocation of STAP-2 to the cell membrane in response
to EGF stimulation also depended on the N-terminal PH domain. In
addition to the membrane-targeting signal, most docking proteins
contain specific domains, such as PTB domains, that are responsible for
complex formation with a particular set of cell surface receptors. For
example, the PTB domains of IRS-1 and IRS-2 bind specifically to
insulin and insulin-like growth factor-1 receptors as well as the IL-4
receptor (31). On the other hand, the PTB domain of FRS2 binds
preferentially to fibroblast growth factor or nerve growth factor
receptors (32). Gab1 and Gab2 contain a Met-binding domain, which can
preferentially interact with c-Met (33, 34). Therefore, each docking
protein may preferentially transmit signals from a specific tyrosine
kinase through its phosphotyrosine-interacting domains. STAP-1 and
STAP-2 also contain an SH2-related domain that may interact with the
phosphotyrosine residues. Because the deletion of the SH2 domain of
STAP-2 resulted in the loss of v-Src-induced STAT3 up-regulation (data
not shown), this SH2-like domain may be responsible for interaction
with tyrosine kinases activated by a particular extracellular stimulus.
Identification of any specific target of the STAP-2 SH2-like domain
will facilitate the understanding of the upstream kinase of STAP-2.
It has been shown that docking proteins function as platforms for the
recruitment of signaling proteins in response to growth factor
stimulation. IRS-1 and IRS-2 amplify the PI3K signal by recruiting PI3K
through their multiple YMXM motifs. Gab1 and FRS2 have been
shown to amplify Ras/MAP kinase signaling by recruiting Grb2 and SHP-2.
We have shown that STAP-2 can potentially associate with PLC Unlike other docking proteins, STAP-2 has additional unique
proline-rich motifs and a YXXQ motif that is a potential
STAT3-binding site in its C terminus. We confirmed the STAP-2 function
of STAT3 activation by overexpression experiments in cell lines.
Furthermore, using knockout mice, we identified the role of STAP-2 in
AP response and STAT3 activation in vivo as well as cultured
hepatocytes in vitro. In AP response, IL-6 has been shown to
play an essential role in hepatocytes (35). The IL-6 receptor
signaling subunit, gp130, is known to elicit the activation of two
major signaling pathways through the activation of JAK kinases: STAT3,
recruited by the YXXQ motif of gp130, and the MAP kinase
pathway, through the SH2-containing protein tyrosine phosphatase
(SHP-2) as a molecular adapter (10). Although both pathways lead to the
activation of transcription factors, on the basis of a number of
in vitro data, STAT3 has been proposed to be the main
mediator of AP gene induction in response to IL-6 (17). However,
because a number of cytokines are induced by LPS, it is not clear
whether IL-6/gp130 is the sole factor for AP gene induction and STAT3
activation during endotoxic shock. For example, in IL-6-deficient mice,
the induction of some AP genes by turpentine treatment was severely impaired, whereas the LPS-induced AP gene induction was normal (36). On
the other hand, conditional deletion of the STAT3 gene in the livers of
adult mice and AP gene induction were defective upon LPS injection
(17). These findings suggest that other factors or molecules in
addition to the IL-6/gp130 system are involved in STAT3 activation for
AP gene induction by LPS. Our STAP-2 knockout mice showed a reduction
in the late phase of STAT3 activation and AP gene indication.
Furthermore, we found that overexpression of STAP-2 in Hep3B cells
enhanced IL-6-induced Although we could not see any potentiation of MAP kinase activation in
response to EGF by STAP-2 overexpression, we could not rule out the
possibility that STAP-2 is involved in signaling pathways other than
STAT3. Our preliminary results indicate instead that STAP-2 inhibits
NF-/
hepatocytes, the
interleukin-6-induced expression of acute-phase (AP) genes and the
tyrosine-phosphorylation level of STAT3 were reduced specifically at
the late phase (6-24 h) of the response. These data indicate that
STAP-2 plays a regulatory role in the AP response in systemic
inflammation. STAP-2 contains a YXXQ motif in the
C-terminal region that is a potential STAT3-binding site. Overexpression of wild-type STAP-2, but not of mutants lacking this
motif, enhanced the AP response element reporter activity and an AP
protein production. These data suggest that STAP-2 is a new
class of adaptor molecule that modulates STAT3 activity through its
YXXQ motif.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(PLC
) are also recruited to receptor tyrosine kinases through their
SH2 domains. Growth factor-induced membrane recruitment of signaling
proteins is also mediated by a family of docking proteins. These
docking proteins contain an N-terminal membrane-targeting domain, such
as the PH domain, and C-terminal multiple tyrosine phosphorylation
sites for recruiting SH2 domain-containing proteins. A significant
effort has been made to search for novel adaptor and docking proteins,
because these molecules will uncover the unique signal transduction and
modulation mechanisms of receptor tyrosine kinases.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PH) (codon 147-403), the SH2-like domain
(
SH2) (lacking 142-242), and the C-terminal region (
C)
(1-242) were also constructed. For GFP fusion, wild-type human STAP-2
and these mutants were subcloned into a pEGFP-C vector (Clontech).
-galactosidase plasmid was co-transfected for each
experiment for the internal control. Cells were grown for 36 h
after transfection, and EGF (100 ng/ml) or LIF (10 ng/ml) was added to
the cells 6 h before harvest. Cell extracts were prepared, and
luciferase and
-galactosidase activities were measured as described
previously (16, 18).
(10 ng/ml, Calbiochem) for the
indicated periods in the presence of cycloheximide (10 µg/ml).
-Fibrinogen and Proinflammatory
Cytokines--
Hep3B stable transfectants were selected and cloned in
the presence of G418 (1 mg/ml). The amount of fibrinogen in the culture supernatant was measured by ELISA (21). The concentration of serum
IL-6, tumor necrosis factor-
, and IL-1
was measured by ELISA
using kits purchased from BIOSOURCE Int. according
to the manufacturer's instructions.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 (23). The C-terminal region of STAP-2
contains several proline-rich motifs (boxed in Fig.
1A) that constitute potential SH3 domain (PXXP) or WW domain
(PXPX) binding sites and four predicted tyrosine
phosphorylation sites (asterisks in Fig. 1A).
Y250EKV is a potential binding site for the SH2 domain of
Src-like kinases as is Y322MNQ for Grb2 and/or STAT3.
Tyr-22 and Tyr-310 are also the potential phosphorylation sites
(asterisk). These motifs are well conserved between human
and mouse (Fig. 1A).
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Fig. 1.
Comparison of the amino acid sequence of STAP
family genes. A, amino acid sequences of STAP-2/BKS and
human STAP-1 (BRDG1). The putative PH domain is marked by a gray
line, and the predicted SH2 domain is indicated with a black
underline. Boxed sequences within the C terminus are
putative SH3 or WW domain-binding sites. The
asterisks indicate the tyrosine phosphorylation sites.
B, schematic view of the domain structures of STAP-2.
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Fig. 2.
Tyrosine phosphorylation and expression of
STAP-2 in cell lines. HEK-293 cells were transiently transfected
with the indicated mutant of Flag-tagged STAP-2 and v-Src
(A-a) or GST-JH1(A-b). Total cell extracts were
immunoblotted with anti-phosphotyrosine ( PY) monoclonal
antibody (4G10) or anti-Flag (
flag). Similar results
were obtained in three independent experiments. B,
endogenous STAP-2 mRNA expression of cell lines. Total RNA was
isolated from indicated cells in exponentially growing state. M1 cells
were cultured in the presence or absence of 10 ng/ml LIF for 3 days.
STAP-2 mRNA was detected by RT-PCR. C-a, parental M1
myeloid cells (STAP-2
) or stable transformants expressing
Myc-tagged wild-type STAP-2 (STAP-2 +) were cultured with 10 ng/ml LIF for 3 days. C-b, Myc-tagged STAP-2
(STAP-2 +) or pcDNA3 vector (STAP-2
) were
transfected transiently to the NIH-3T3 cells that stably express EGF
receptor. Cells were stimulated with 100 ng/ml EGF for 10 min. In each
experiment, total cell lysates were immunoblotted with
anti-phosphotyrosine (
PY) monoclonal antibody (4G10) or
anti-Myc (
Myc) monoclonal antibody (9E10). The
black arrowheads indicate tyrosine-phosphorylated
STAP-2. The white arrowheads indicate the LIF receptor
(C-a) and the EGF receptor (C-b).
PH) did
not translocate to the plasma membrane (Fig. 3B). A GFP
fused to the PH domain alone was translocated to the plasma membrane in
response to EGF (data not shown). Therefore, the PH domain of STAP-2 is
necessary and sufficient for plasma membrane recruitment in response to
EGF.
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Fig. 3.
EGF-induced translocation of STAP-2.
A431 cells were transiently transfected with GFP-fused wild-type
STAP-2 (WT-GFP) (A) or a deletion mutant lacking
the PH domain of STAP-2 ( PH-GFP) (B). 36 h after transfection, cells were stimulated with EGF (100 ng/ml) for
the indicated periods. Then each sample was fixed, and fluorescence
images were obtained by laser confocal microscopy (Pascal, Carl
Zeiss).
B, and STATs within the 2-kb of the 5' flanking region of the
STAT-2 transcription initiation sites (data not shown). Because these
transcription factors are often activated by bacterial pathogens and
inflammatory cytokines including IL-1, tumor necrosis factor-
, and
IL-6, we examined the effect of intraperitoneal injection of LPS on the
STAP-2 mRNA levels in mouse liver. RT-PCR analysis with specific
primers for STAP-2 revealed that STAP-2 expression was markedly
elevated at 3 to 6 h after LPS challenge in mouse liver (Fig.
4A). To determine which
stimuli directly induce STAP-2 expression, we examined the induction of
STAP-2 in the primary hepatocytes or macrophages in the presence of
cycloheximide. As shown in Fig. 4B, STAP-2 mRNA levels
were elevated in hepatocytes, but not in macrophages, by IL-6 and
IL-1
but not by LPS (Fig. 4B). We confirmed the
IL-6-induced up-regulation of STAP-2 mRNA levels by Northern
hybridization analysis in primary hepatocytes (Fig. 4C-b) as
well as the Hep3B human hepatoma cell line (Fig. 4C-b).
These data indicate that STAP-2 expression is regulated by several
inflammatory cytokines, including IL-6, in hepatocytes. Thus, STAP-2
could mediate IL-6 signaling in acute-phase (AP) response.
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Fig. 4.
Induction of STAP-2 by inflammatory
cytokines. A, transient expression of STAP-2 mRNA
in the liver after LPS injection into mice. The liver of C57BL/6 mice
after intraperitoneal (i.p.) injection of LPS was isolated
at the indicated periods, and STAP-2 mRNA levels were determined by
RT-PCR analysis. Similar results were obtained in three independent
experiments. B and C, induction of STAP-2
expression in primary hepatocytes (B-a) or macrophages
(B-b) isolated from C57BL/6 mice. Cells were stimulated with
LPS, IL-6, or IL-1 at the indicated periods in the presence of
cycloheximide (10 µg/ml) after 12 h serum starvation. STAP-2
mRNA levels were determined by RT-PCR analysis. C,
Northern blot analysis of primary hepatocytes (C-a) or Hep3B
cells (C-b) for STAP-2 mRNA induction. Cells were
stimulated with 10 ng/ml IL-6 for the indicated periods in the presence
of cycloheximide (10 µg/ml).
/
mice appeared to be healthy and showed no apparent abnormalities in
most organs, including the digestive and respiratory organs, heart,
blood vessels, muscles, and lymphoid tissues, at the gross and
histological levels (data not shown).
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Fig. 5.
Generation of STAP-2-deficient mice.
A, schematic representations of wild-type and mutant loci of
the STAP-2 gene together with the targeting vector. Exons for genes
encoding STAP-2 are represented by black boxes. The neomycin
resistance gene, driven by a PGK promoter (pgk-neo), and the
gene coding the thymidine kinase, driven by an HSV promoter
(hsv-tk), are indicated by white boxes.
EcoRV-digested genomic DNA fragments were detected by probe.
Ev, EcoRV restriction site. B,
representative Southern blot analysis with EcoRV-digested
DNA. Of 233 offspring from crosses between F2 heterozygous mice, 60 were STAP-2+/+, 119 were STAP-2+/ , and 54 were STAP-2
/
. C, Northern blot analysis of
total RNA from wild-type and STAP-2+/
and
STAP-2
/
mice showed no STAP-2 mRNA expression.
/
mice were
treated once with LPS. Liver samples were then collected at different
times, and the mRNA levels of AP genes, SAP, and haptoglobin were
measured by Northern hybridization analysis (Fig. 6A). The haptoglobin (Fig.
6A-a) and SAP (Fig. 6A-b) mRNA levels normalized to GAPDH mRNA are also plotted on graphs. Induction of
these two AP genes, especially SAP, in liver was reduced in STAP-2
/
mice after the LPS challenge. However, IL-6,
IL-1
, and tumor necrosis factor-
levels induced by LPS mostly
from macrophages were not different between STAP-2
/
mice and their wild-type littermates (data not shown). These data
suggest that the STAP-2 protein may positively regulate AP gene
induction in hepatocytes.
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Fig. 6.
AP protein induction and STAT3 activation in
response to LPS in STAP-2 /
mice or in response to IL-6 in STAP-2-deficient hepatocytes.
A, induction of haptoglobin and SAP mRNAs by LPS was
measured by Northern blot analysis on STAP-2
/
mice and
STAP-2+/+ littermates. The mice were injected with either
LPS or saline solution, and their livers were collected after 0, 3, 6, and 24 h. 20 µg of total RNA for each sample was analyzed. The
relative intensities of SAP/GAPDH or haptoglobin/GAPDH are shown as
mean values with standard errors from at least three mice/group in the
upper panels. One representative Northern blotting data from
each experiment is shown in the lower panels.
B, Northern blot analysis of IL-6-induced SAP transcription
of the wild-type and STAP-2
/
hepatocytes. Relative
band-intensities of SAP/GAPDH of the left panel are shown in the
right panel. Similar results were obtained in two
independent experiments. C, panels a and
b, total cell lysates from liver of the LPS-injected
mice or IL-6-stimulated primary hepatocytes were
immunoblotted with anti-STAT3 (
STAT3)- or
anti-phosphotyrosine STAT3 (
PY-STAT3)-specific
antibodies. The lower panels show the relative band
intensities of PY-STAT3/STAT3 of Western blotting. Representative data
from two independent experiments are shown.
/
cultured
hepatocytes. However, after 6 h, the SAP mRNA level decreased
in STAP-2
/
hepatocytes but was elevated in
STAP-2+/+ hepatocytes. This late effect of STAP-2
deficiency on IL-6-induced SAP induction is consistent with the time
course of induction of STAP-2 by IL-6 (Fig. 4).
PH) in MCF7 cells. The
STAP-2Y22F mutant instead suppressed STAT3 activation,
suggesting that this mutant function was a dominant negative form,
because a high level of endogenous STAP-2 protein was detected by
Western blotting in MCF7 cells (12).
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Fig. 7.
STAP-2 potentiates STAT3 activity in the cell
lines. A, APRE-luciferase activity was measured in
HEK-293 cells transiently transfected with the indicated dose of a
wild-type STAP-2 construct with the APRE-luciferase gene (0.2 µg),
the EGF receptor, and the -galactosidase plasmid (0.05 µg) as an
internal control. Cells were stimulated with either EGF or LIF for
6 h, and cell extracts were prepared. B, Elk-1 reporter
gene assay. HEK-293 cells were transfected with the indicated amount of
STAP-2, the Elk-1-luciferase gene (0.2 µg), the EGF receptor (0.1 µg), and the
-galactosidase plasmid (0.05 µg). Cells were
stimulated with EGF for 6h. C, APRE-luciferase activity was
measured in MCF-7 cells after transfection with the indicated STAP-2
mutant constructs (0.1 µg) in the presence or absence of v-Src (0.1 µg). The mean values and standard deviations from three independent
experiments are shown. D, IL-6-induced
-fibrinogen
production from Hep3B cells stably expressing wild-type (WT)
STAP-2 and deletion mutants lacking the PH domain (
PH),
the SH2-like domain (
SH2), and the C-terminal region
(
CH). Cells were stimulated with IL-6 for 48 h, and
the amount of
-fibrinogen secreted into the medium was
measured by ELISA. E, association of STAP-2 with
STAT3. Wild-type STAP-2 (WT), STAP-2Y250F
(Y250F), STAP-2Y322F (Y322F), and
pcDNA3 (vector) were transfected into 293 cells (1 × 106 cells) with v-Src, and the lysates were
immunoprecipitated with anti-STAT3 antibody. The total cell lysate
(TCL) or immunoprecipitates (IP) were
immunoblotted (IB) with anti-Flag or anti-STAT3.
-fibrinogen, one of the AP proteins, by ELISA. Wild-type STAP-2, but
not deletion mutants, strongly enhanced IL-6-induced
-fibrinogen production in Hep3B cells. These data suggest that overexpression of
STAP-2 potentiates cytokine- as well as tyrosine
kinase-dependent STAT3 activation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
liver or hepatocytes in response to
endotoxin shock or IL-6, respectively. These data indicate that STAP-2
is a novel class of adaptor/docking molecules that potentiate STAT3
activation in response to IL-6-related cytokines or other stimuli.
1 and
Grb2; however, STAP-2 had no effect on the Ras/MAP kinase pathway
induced by EGF (Fig. 6B) or on intracellular calcium signaling.2
-fibrinogen production and that STAP-2 was
phosphorylated in LIF-treated M1 cells. Thus, STAP-2 seems to be one of
the molecules that supports or potentiates STAT3 activation.
Understanding the precise mechanism of STAT3 activation through STAP-2
may be important for the control of the AP response and bacterial infection.
B transcriptional activation. Because STAP-2 expression is
induced by proinflammatory cytokines such as IL-6 or IL-1
, STAP-2
may play a role in the regulation of inflammation. The STAP-2 gene
knockout mice will provide a powerful tool for such investigations.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Ohgusu, M. Sasaki, and I. Ueno for their excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Ministry of Education, Science, Technology, Sports, and Culture of Japan, the Japan Health Science Foundation, the Human Frontier Science Program, the Japan Research Foundation for Clinical Pharmacology, and the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to GenBankTM/EBI Data Bank with the accession number(s) AW049765.
¶ To whom correspondence should be addressed: Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan. Fax: 81-92-642-6825; E-mail: yakihiko@bioreg.kyushu-u.ac.jp.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M211230200
2 M. Minoguchi, S. Minoguchi, D. Aki, A. Joo, T. Yamamoto, T. Yumioka, T. Matsuda, and A. Yoshimura, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
SH2 and -3, Src
homology-2 and -3;
STAP, signal-transducing adaptor protein;
MAP, mitogen-activated protein;
PH, pleckstrin homology;
PI3K, phosphoinositide-3-OH kinase;
PLC, phospholipase C
;
STAT, signal
transducers and activators of transcription;
JAK, Janus kinase;
IL, interleukin;
EGF, epidermal growth factor;
LIF, leukemia inhibitory
factor;
AP, acute phase;
GST, glutathione S-transferase;
LPS, lipopolysaccharide;
SAP, serum amyloid P;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GFP, green fluorescent
protein;
APRE, acute-phase response element;
ELISA, enzyme-linked
immunosorbent assay;
RACE, rapid amplification of cDNA ends;
BRK, breast tumor kinase;
PTB, phosphotyrosine-binding domain;
IRS, insulin
receptor substrate.
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