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• W2062087727 abstract "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. 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. Src homology-2 and -3 signal-transducing adaptor protein mitogen-activated protein pleckstrin homology phosphoinositide-3-OH kinase phospholipase Cγ signal transducers and activators of transcription Janus kinase interleukin epidermal growth factor leukemia inhibitory factor acute phase glutathione S-transferase lipopolysaccharide serum amyloid P glyceraldehyde-3-phosphate dehydrogenase green fluorescent protein acute-phase response element enzyme-linked immunosorbent assay rapid amplification of cDNA ends breast tumor kinase phosphotyrosine-binding domain insulin receptor substrate 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 (1Stanley E.R. Guilbert L.J. Tushinski R.J. Bartelmez S.H. J. Cell. Biochem. 1983; 21: 151-159Google Scholar, 2Rohrschneider L.R. Guidebook to Cytokines and Their Receptors. Oxford University Press, Oxford, UK1995: 168-170Google Scholar). 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 Cγ (PLCγ) 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 (3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Google Scholar). These proteins become activated through tyrosine phosphorylation, which typically occurs through cytokine receptor-associated kinases, the Janus kinase (JAK) family proteins (5Ihle J.N. Nature. 1995; 377: 591-594Google Scholar). 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 (6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Google Scholar), and we recently reported that STAT3 is activated by the hepatitis type C virus core protein (7Yoshida T. Hanada T. Tokuhisa T. Kosai K. Sata M. Kohara M. Yoshimura A. J. Exp. Med. 2002; 196: 641-653Google Scholar). Usually, STAT3 is activated by IL-6-related cytokines, IL-10 and the granulocyte colony-stimulating factor (4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Google Scholar). Moreover, STAT 3 was also activated by many tyrosine kinases unrelated to those cytokines, including v-Src, the EGF receptor, and c-Kit (8Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 5: 2553-2558Google Scholar, 9Turkson J. Bowman T. Garcia R. Caldenhoven E. De Groot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Google Scholar). Cytokine receptors that activate STAT3 possess a YXXQ motif in the cytoplasmic region, which recruits STAT3 to the receptor (10Hirano T. Ishihara K. Hibi M. Oncogene. 2000; 19: 2548-2556Google Scholar). 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 (11Besser D. Bromberg J.F. Darnell J.E. Hanafusa H. Mol. Cell. Biol. 1999; 19: 1401-1409Google Scholar). 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 (12Mitchell P.J. Sara E.A. Crompton M.R. Oncogene. 2000; 19: 4273-4282Google Scholar). 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. The cytoplasmic domain of c-fms (codon 560–926) was inserted in-frame into the GAL4 DNA-binding domain vector pBTM116 (13Yokouchi M. Suzuki R. Masuhara M. Komiya S. Inoue A. Yoshimura A. Oncogene. 1997; 15: 7-15Google Scholar). The fetal liver yeast two-hybrid cDNA library in pACT2 was purchased from Clontech. More than 2 × 106transformants were screened according to the procedure described previously (14Masuhara M. Nagao K. Nishikawa M. Sasaki M. Yoshimura A. Osawa M. Biochem. Biophys. Res. Commun. 2000; 268: 697-703Google Scholar). 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.4 A). The hsv-tkgene was inserted for negative selection. Homologous recombination in murine embryonic stem cells was performed as described previously (15Ohtsuka S. Takaki S. Iseki M. Miyoshi K. Nakagata N. Kataoka Y. Yoshida N. Takatsu K. Yoshimura A. Mol. Cell. Biol. 2002; 22: 3066-3077Google Scholar) 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. 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. 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 mNaCl, 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′. 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 (16Hanada T. Yoshida T. Kinjyo I. Minoguchi S. Yasukawa H. Kato S. Mimata H. Nomura Y. Seki Y. Kubo M. Yoshimura A. J. Biol. Chem. 2001; 276: 40746-40754Google Scholar). Total proteins from mouse liver were prepared as described previously (17Alonzi T. Maritano D. Gorgoni B. Rizzuto G. Libert C. Poli V. Mol. Cell. Biol. 2001; 21: 1621-1632Google Scholar). 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 (ΔPH) (codon 147–403), the SH2-like domain (ΔSH2) (lacking 142–242), and the C-terminal region (ΔC) (1–242) were also constructed. For GFP fusion, wild-type human STAP-2 and these mutants were subcloned into a pEGFP-C vector (Clontech). 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) (16Hanada T. Yoshida T. Kinjyo I. Minoguchi S. Yasukawa H. Kato S. Mimata H. Nomura Y. Seki Y. Kubo M. Yoshimura A. J. Biol. Chem. 2001; 276: 40746-40754Google Scholar, 18Sasaki A. Taketomi T. Wakioka T. Kato R. Yoshimura A. J. Biol. Chem. 2001; 276: 36804-36808Google Scholar) 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 β-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 β-galactosidase activities were measured as described previously (16Hanada T. Yoshida T. Kinjyo I. Minoguchi S. Yasukawa H. Kato S. Mimata H. Nomura Y. Seki Y. Kubo M. Yoshimura A. J. Biol. Chem. 2001; 276: 40746-40754Google Scholar, 18Sasaki A. Taketomi T. Wakioka T. Kato R. Yoshimura A. J. Biol. Chem. 2001; 276: 36804-36808Google Scholar). Primary hepatocytes were prepared using the two-step collagenase perfusion method as described (19Brysha M. Zhang J.G. Bertolino P. Corbin J.E. Alexander W.S. Nicola N.A. Hilton D.J. Starr R. J. Biol. Chem. 2001; 276: 22086-22089Google Scholar). After isolation, the cells were washed two times in Williams' E medium with 10% fetal bovine serum, l-glutamine (Sigma), 100 nmdexamethasone (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% CO2at 37 °C for 36 h prior to the experiments. Intraperitoneal macrophages were prepared and cultured as described previously (20Kinjyo I. Hanada T. Inagaki-Ohara K. Mori H. Aki D. Ohishi M. Yoshida H. Kubo M. Yoshimura A. Immunity. 2002; 17: 583-591Google Scholar). For each experiment, cells were stimulated with LPS (10 ng/ml) (Sigma), IL-6 (50 ng/ml, Calbiochem), or IL-1β (10 ng/ml, Calbiochem) for the indicated periods in the presence of cycloheximide (10 μg/ml). 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 (21Natsuka S. Isshiki H. Akira S. Kishimoto T. FEBS Lett. 1991; 291: 58-62Google Scholar). The concentration of serum IL-6, tumor necrosis factor-α, and IL-1β was measured by ELISA using kits purchased from BIOSOURCE Int. according to the manufacturer's instructions. The nucleotide sequence reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases under accession numberAW049765. 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 (12Mitchell P.J. Sara E.A. Crompton M.R. Oncogene. 2000; 19: 4273-4282Google Scholar). The mouse STAP-2 gene encodes a protein of 411 amino acids that is structurally related to STAP-1 (stem cell-specificadaptor protein-1) (14Masuhara M. Nagao K. Nishikawa M. Sasaki M. Yoshimura A. Osawa M. Biochem. Biophys. Res. Commun. 2000; 268: 697-703Google Scholar). 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 (22Ohya K. Kajigaya S. Kitanaka A. Yoshida K. Miyazato A. Yamashita Y. Yamanaka T. Ikeda U. Shimada K. Ozawa K. Mano H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11976-11981Google Scholar). Because STAP-2 is expressed in a variety of tissues, we changed the definition of STAP to signal-transducingadaptor 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 PLCγ2 (23Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Google Scholar). The C-terminal region of STAP-2 contains several proline-rich motifs (boxed in Fig.1 A) 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. 1 A). 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. 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.2 A-a), a JAK2 tyrosine kinase domain fused to GST (GST-JH1) (Fig. 2 A-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.2 A, 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. 2 B). 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.2 C-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. 2 C-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.3 A, 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. 3 A). The mutant STAP-2 lacking an N-terminal PH domain (Δ PH) did not translocate to the plasma membrane (Fig. 3 B). 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. Although STAP-2 has been shown to be relatively widely expressed in murine and human tissues (data not shown and Ref. 12Mitchell P.J. Sara E.A. Crompton M.R. Oncogene. 2000; 19: 4273-4282Google Scholar), 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-κB, 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-α, 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.4 A). 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. 4 B, STAP-2 mRNA levels were elevated in hepatocytes, but not in macrophages, by IL-6 and IL-1β but not by LPS (Fig. 4 B). We confirmed the IL-6-induced up-regulation of STAP-2 mRNA levels by Northern hybridization analysis in primary hepatocytes (Fig. 4 C-b) as well as the Hep3B human hepatoma cell line (Fig. 4 C-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. 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.5 A). The disruption of STAP-2 gene and expression were confirmed by Southern blot analysis (Fig.5 B) and Northern blot analysis of total RNA from the heart (Fig. 5 C), 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). 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.6 A). The haptoglobin (Fig.6 A-a) and SAP (Fig. 6 A-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-1β, and tumor necrosis factor-α 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. 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 (35Fattori E. Cappelletti M. Costa P. Sellitto C. Cantoni L. Carelli M. Faggioni R. Fantuzzi G. Ghezzi P. Poli V. J. Exp. Med. 1994; 180: 1243-1250Google Scholar), inducing STAP-2 mRNA expression by IL-6 in hepatocytes (see Fig.4 B). As shown in Fig. 6 B, 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 (17Alonzi T. Maritano D. Gorgoni B. Rizzuto G. Libert C. Poli V. Mol. Cell. Biol. 2001; 21: 1621-1632Google Scholar). 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. 6 C-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. 6 C-b). In the mutant hepatocytes, the re-up-regulation after 6 h was not observed (Fig.6 C-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. 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 (10Hirano T. Ishihara K. Hibi M. Oncogene. 2000; 19: 2548-2556Google Scholar, 24Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka A. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Google Scholar). Wild-type STAP-2 enhanced EGF- or LIF-induced APRE-reporter activation in HEK-293 cells (Fig. 7 A). With the same conditions, the Elk-1 reporter gene, which reflects MAP kinase activation (18Sasaki A. Taketomi T. Wakioka T. Kato R. Yoshimura A. J. Biol. Chem. 2001; 276: 36804-36808Google Scholar, 25Wakioka T. Sasaki A. Kato R. Shouda T. Matsumoto A. Miyoshi K. Tsuneoka M. Komiya S. Baron R. Yoshimura A. Nature. 2001; 412: 647-651Google Scholar), was not enhanced by STAP-2 (Fig. 7 B). As shown in Fig. 7 C, 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 (ΔPH) 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 (12Mitchell P.J. Sara E.A. Crompton M.R. Oncogene. 2000; 19: 4273-4282Google Scholar). We next examined the effect of STAP-2 overexpression on IL-6-induced AP protein in a hepatoma cell line (Fig. 7 D). We measured β-fibrinogen, one of the AP proteins, by ELISA. Wild-type STAP-2, but not deletion mutants, strongly enhanced IL-6-induced β-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. 7 E, 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 (14Masuhara M. Nagao K. Nishikawa M. Sasaki M. Yoshimura A. Osawa M. Biochem. Biophys. Res. Commun. 2000; 268: 697-703Google Scholar). STAP-1 also was identified as a Tec-interacting protein that is tyrosine-phosphorylated in response to B-cell receptor stimulation, termed BRDG1 (22Ohya K. Kajigaya S. Kitanaka A. Yoshida K. Miyazato A. Yamashita Y. Yamanaka T. Ikeda U. Shimada K. Ozawa K. Mano H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11976-11981Google Scholar). 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 (26Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Google Scholar, 27Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Google Scholar, 28Grimm J. Sachs M. Britsch S. Di Cesare S. Schwarz-Romond T. Alitalo K. Birchmeier W. J. Cell Biol. 2001; 154: 345-354Google Scholar). Some docking proteins are associated with the cell membrane by a myristyl anchor (e.g. FRS2) or a transmembrane domain (e.g. LAT) (29Burack W.R. Cheng A.M. Shaw A.S. Curr. Opin. Immunol. 2002; 14: 312-316Google Scholar). 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 (30Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Google Scholar). 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 (31Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Google Scholar). On the other hand, the PTB domain of FRS2 binds preferentially to fibroblast growth factor or nerve growth factor receptors (32Ong S.H. Guy G.R. Hadari Y.R. Laks S. Gotoh N. Schlessinger J. Lax I. Mol. Cell. Biol. 2000; 20: 979-989Google Scholar). Gab1 and Gab2 contain a Met-binding domain, which can preferentially interact with c-Met (33Lock L.S. Maroun C.R. Naujokas M.A. Park M. Mol. Biol. Cell. 2002; 13: 2132-2146Google Scholar, 34Bardelli A. Longati P. Gramaglia D. Stella M.C. Comoglio P.M. Oncogene. 1997; 15: 3103-3111Google Scholar). 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 PLCγ1 and Grb2; however, STAP-2 had no effect on the Ras/MAP kinase pathway induced by EGF (Fig. 6 B) or on intracellular calcium signaling. 2M. Minoguchi, S. Minoguchi, D. Aki, A. Joo, T. Yamamoto, T. Yumioka, T. Matsuda, and A. Yoshimura, unpublished data. 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 (35Fattori E. Cappelletti M. Costa P. Sellitto C. Cantoni L. Carelli M. Faggioni R. Fantuzzi G. Ghezzi P. Poli V. J. Exp. Med. 1994; 180: 1243-1250Google Scholar). 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 (10Hirano T. Ishihara K. Hibi M. Oncogene. 2000; 19: 2548-2556Google Scholar). Although both pathways lead to the activation of transcription factors, on the basis of a number ofin vitro data, STAT3 has been proposed to be the main mediator of AP gene induction in response to IL-6 (17Alonzi T. Maritano D. Gorgoni B. Rizzuto G. Libert C. Poli V. Mol. Cell. Biol. 2001; 21: 1621-1632Google Scholar). 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 (36Alonzi T. Fattori E. Cappelletti M. Ciliberto G. Poli V. Cytokine. 1998; 10: 13-18Google Scholar). 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 (17Alonzi T. Maritano D. Gorgoni B. Rizzuto G. Libert C. Poli V. Mol. Cell. Biol. 2001; 21: 1621-1632Google Scholar). 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 β-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-κB transcriptional activation. Because STAP-2 expression is induced by proinflammatory cytokines such as IL-6 or IL-1β, 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. We thank H. Ohgusu, M. Sasaki, and I. Ueno for their excellent technical assistance." @default.
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• W2062087727 title "STAP-2/BKS, an Adaptor/Docking Protein, Modulates STAT3 Activation in Acute-phase Response through Its YXXQ Motif" @default.
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