STAP-2/BKS, an Adaptor/Docking Protein, Modulates STAT3 Activation in Acute-phase Response through Its YXXQ Motif*

Mayu MinoguchiDagger , Shigeru MinoguchiDagger , Daisuke AkiDagger , Akiko JooDagger , Tetsuya Yamamoto§, Taro Yumioka§, Tadashi Matsuda§, and Akihiko YoshimuraDagger

From the Dagger  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

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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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-/- 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.

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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 Cgamma (PLCgamma ) 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.

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.

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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 (Delta PH) (codon 147-403), the SH2-like domain (Delta SH2) (lacking 142-242), and the C-terminal region (Delta 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).

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 beta -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 beta -galactosidase activities were measured as described previously (16, 18).

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-1beta (10 ng/ml, Calbiochem) for the indicated periods in the presence of cycloheximide (10 µg/ml).

Measurement of beta -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-alpha , and IL-1beta was measured by ELISA using kits purchased from BIOSOURCE Int. according to the manufacturer's instructions.

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.

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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 PLCgamma 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.

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).


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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 (alpha  PY) monoclonal antibody (4G10) or anti-Flag (alpha  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 (alpha  PY) monoclonal antibody (4G10) or anti-Myc (alpha  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).

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 (Delta 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 (Delta 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).

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-kappa 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-alpha , 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-1beta 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-1beta 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).

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-/- 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.

We then investigated the effect of STAP-2 deficiency on AP response in the liver. STAP-2+/+ and STAP-2-/- 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-1beta , and tumor necrosis factor-alpha 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 (alpha  STAT3)- or anti-phosphotyrosine STAT3 (alpha  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.

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-/- 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).

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 (Delta 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 beta -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 beta -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 beta -fibrinogen production from Hep3B cells stably expressing wild-type (WT) STAP-2 and deletion mutants lacking the PH domain (Delta PH), the SH2-like domain (Delta SH2), and the C-terminal region (Delta CH). Cells were stimulated with IL-6 for 48 h, and the amount of beta -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.

We next examined the effect of STAP-2 overexpression on IL-6-induced AP protein in a hepatoma cell line (Fig. 7D). We measured beta -fibrinogen, one of the AP proteins, by ELISA. Wild-type STAP-2, but not deletion mutants, strongly enhanced IL-6-induced beta -fibrinogen production in Hep3B cells. These data suggest that overexpression of STAP-2 potentiates cytokine- as well as tyrosine kinase-dependent STAT3 activation.

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.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-/- 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.

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 PLCgamma 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

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 beta -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.

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-kappa B transcriptional activation. Because STAP-2 expression is induced by proinflammatory cytokines such as IL-6 or IL-1beta , 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.

    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; PLCgamma , phospholipase Cgamma ; 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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