論文

JAB/suppressor of cytokine signaling 1 (SOCS1) STAT-induced STAT inhibitor-1 (SSI-1) (JAB/SOCS1/SSI-1) is an SH2-domain-containing protein that is induced by and negatively regulates signaling by a number of cytokines including interleukin-4 (IL-4), IL-6, interferon (IFN)-γ, prolactin, growth hormone, and erythropoietin. The role of JAB/SOCS1/SSI-1 in IL-2 signaling has been analyzed. JAB/SOCS1/SSI-1 is strongly induced by IL-2 in peripheral blood T cells, and JAB/SOCS1/SSI-1 overexpression strongly inhibits IL-2-induced signal transducer and activator of transcription-5 (Stat5) phosphorylation and transcriptional activity. In cotransfection experiments, JAB/SOCS1/SSI-1 associates with both Jak1 and Jak3; however, JAB/SOCS1/SSI-1 had a greater effect on Jak1 tyrosine phosphorylation and kinase activity. JAB/SOCS1/SSI-1 also interacts with IL-2Rβ, and this interaction requires the A region (residues 313-382) of IL-2Rβ. However, this interaction was not essential for the inhibitory action of JAB. Thus, JAB/SOCS1/SSI-1 is an IL-2-induced inhibitor of IL-2 signaling that functions by inhibiting Jak kinase activity. This suggests an important role for JAB/SOCS1/SSI-1 in regulating T-cell responses. © 2001 by The American Society of Hematology.

The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Among them, we have shown that JAB/SOCS-1 is strongly induced by interferon-γ and forced expression of JAB/SOCS-1 conferred cells interferon resitance. This resistance was caused by inhibition of JAK1 and JAK2 activation in response to lFNγ. Moreover, recent detailed analysis of JAB/SOCS-1 knockout mice revealed that JAB/SOCS-1 is indeed a 'negative feedback regulator' that determine the sensitivity of cells to IFNγ. Using in vitro mutagensis, we defined a functional structure of JAB/SOCS-1 and proposed a mechanism for how JAB inhibits JAK kinase activity.

The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred to as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Most of them appear to be induced after stimulation with several different cytokines, and at least three of them (CIS 1, CIS3/SOCS3, and JAB/SOCS 1) negatively regulate cytokine signal transduction by various means: CIS 1 inhibits STAT5 activation by binding to cytokine receptors that recruit STAT5, whereas JAB/SOCS-1 and CIS3/SOCS-3 directly bind to the kinase domain of JAKs, thereby inhibiting tyrosine-kinase activity. Therefore, these CIS family members seem to function in a classical negative feedback loop of cytokine signaling. Biochemical characterization as well as gene disruption studies indicate that JAB/SOCS-1/SSI-1 is an important negative regulator of interferon γ signaling. The mechanisms by which these inhibitors of cytokine signal transduction exert their effects have been extensively studied and will provide useful information for regulating tyrosine-kinase activity.

Interleukin 13 (IL-13) has been shown to play a critical role in the allergic diseases and bronchial asthma. However, signal transduction mechanisms for IL-13 receptor complex is still completely not clear. To investigate its signal transduction pathways, we have cloned new IL-13Ralpha' binding protein by yeast tri-hybrid system and designated it interleukin 13 receptor binding protein 1 (IL-I3RBP1). IL-13RBP1 cDNA is 2370 bp in length with open reading frame predicted to encode a protein of 625 amino acids. MOTIF search analysis revealed that IL-13RBP1 cDNA contains a myosin heavy chain like sequence in the N terminal end, nuclear localization signal in the middle part and coiled-coil region in C terminal end. We next examined mRNA expression in human tissues by Northern analysis and found that two different sizes (4.4 and 2.4 kb) of IL13RBP1 transcripts were present in most tissues. In addition, testis contained an additional 3 types of transcripts of -2.7 kb. Database searches utilizing BLAST and FASTA programs failed to identify any mammalian proteins having significant sequence similarities to IL13RBP1. However, C. elegans and D. melanogaster proteins of unknown function share -20% similarity to IL-13RBP1. IL13RBP1 may provide a novel mechanism in IL-13R signal transduction pathways.

Receptor activation by the haematopoietic growth factor proteins interleukin 5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) leads to phosphorylation of JAK2 as a key trigger of signal transduction. JAB has recently been identified as a regulator of JAK2 phosphorylation and activity by binding phosphorylated JAK2 and inducing its degradation. As part of our effort to define molecular recognition networks that lead to signalling, we investigated the effect of JAB on both JAK2 phosphorylation and JAK2 interaction state that ensue upon IL-5 stimulation in recombinant 293T cells cotransfected 293T cells with IL-5Rα, βc and hJAK2 either with or without JAB. Without JAB, stimulation with wild-type and re-engineered single chain (sc) IL-5 induced a time-dependent phosphorylation of JAK2. In the presence of JAB cotransfection, no phospho-JAK2 was observed, and JAB was observed co-immunoprecipitated with non-phosphorylated JAK2. The time dependence of JAB co-immunoprecipitation correlated with the time dependence of JAK2 phosphorylation when JAB was absent. Since JAB has already been shown to bind JAK2 via a phosphorylated tyrosine, the current data suggest that JAB binds to phosphorylated JAK2, enhances JAK2 dephosphorylation and remains associated in a complex, with dephosphorylated JAK2, that may be a precursor leading to irreversible JAK2 degradation. (C) 2000 Academic Press.

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.

Recently, constitutive activation of JAK kinases (JAKs) and/or signal transducers and activators of transcription (STATs) has been reported in growing numbers of human cancer cells as well as oncogene-transformed cells. JAB/SOCS-1 has been shown to be an intrinsic JAK tyrosine kinase inhibitor and to suppress the cytokine-dependent JAK-STAT pathway. In this report, we investigated the effect of ectopic expression of JAB on v-Src-induced JAK-STAT activation. Forced expression of JAB in v-Src-transformed NIH3T3 cells neither suppressed phosphorylation of STAT3 and JAK1/JAK2 nor blocked STAT3-reporter gene activation. Colony forming assay also showed that JAB did not suppress v-Src-lnduced transformation of NIH3T3 cells, while dominant negative STAT3 suppressed it. In contrast, JAB could downregulate pbospborylation of STAT1 and STAT3 induced by interferon gamma (IFN(γ)) and interleukin-6 (IL-6) plus soluble IL6 receptor (sIL-6R), respectively. Furthermore, in vitro kinase assay indicated that JAB suppressed byperactivation of JAK1/JAK2 and JAK1 induced by INF(γ) and IL-6 plus sIL-6R respectively, but not v-Src-induced basal JAKI/JAK2 activity. Nevertheless, both JAK1/JAK2 activated by v-Src and that activated by IL-6 plus slL-6R could similarly bind JAB. These results clearly demonstrate that JAB distinguishes cytokine-induced JAK-STAT signaling from v-Src-induced one and can not suppress the transformation with v-Src.

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr401 in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr401 region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly45 was critical for binding to the EPOR but not to JAK2, while Leu22 was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr401, reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an on-cogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185(c)-(erbB)-2. Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.

In cultured vascular smooth muscle cells (VSMCs), Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are expressed constitutively and play a role in angiotensin II (Ang II)-induced intracellular signaling and proliferation. However, little is known regarding the relevance of these proteins to the process of vascular remodeling. The role of JAK and STAT proteins in vascular remodeling and their functional coupling with Ang II were examined in balloon-injured rat carotid artery. Immunoreactive Jak2, Tyk2, Stat1, and Stat3 were not detected in the intact artery. Immunohistostaining showed transient expressions of these JAKs and STATs in medial and neointimal VSMCs at days 2 and 5, respectively, with a peak at day 7 in both layers. The expressions declined to insignificant levels by day 14. Ang II type 1 receptors (AT1s) were coexpressed in the medial and neointimal VSMCs expressing Jak2 and Stat3. The Jak2 and Stat3 inductions in the injured artery were accompanied by constitutive Jak2 and Stat3 phosphorylations, which were enhanced by ex vivo Ang II stimulation via AT1. Additionally, a Jak2 inhibitor, AG490, blocked the Ang II-induced Stat3 phosphorylation. Furthermore, local treatment with AG490 inhibited constitutive Stat3 phosphorylation and neointimal VSMC replication and subsequently reduced neointima formation in the injured artery. In conclusion, JAK and STAT proteins were inducible in medial and neointimal VSMCs after vascular injury and were functionally coupled to AT1. The inductions of JAKs and STATs would be involved in the mechanisms of neointima formation after vascular injury.

To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T. We demonstrated that co-expression of CD8/ζ and Syk were necessary for NF-AT activation in 293T. This NF-AT response was completely inhibited by the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tec protein tyrosine kinase. We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/ζ. These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3ζ/Syk-mediated NF-AT activation. Copyright (C) 2000 Federation of European Biochemical Societies.

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1+c-kit+Lin-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1+c-kit+Lin- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.

Crosslinking of multivalent antigen bound IgE transduces FcεRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcεRI-mediated calcium mobilization and degranulation. FcεRI-induced tyrosine phosphorylation of phospholipase C-γ (PLC-γ) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-γ in vivo. These data indicate that STAP-2/BSK negatively controls the FcεRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-γ. © 2003 Elsevier Science (USA). All rights reserved.

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/ or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.

Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling.

WSX-1 is a member of the class I cytokine receptor family with homology to IL-12Rβ2 and is essential for the initial mounting of Th1 responses. STAT1 interacts with tyrosine-phosphorylated WSX-1, and the conserved tyrosine residue of the cytoplasmic domain of WSX-1 is essential for transcriptional activation of STAT1. IL-27 stimulation induced STAT1 phosphorylation in wild-type but not in WSX-1-deficient naive CD4+ T cells. Although IL-27 did not directly induce IFN-γ production by wild-type CD4+ T cells, IL-12-dependent IFN-γ production was augmented by IL-27 stimulation in wild-type naive CD4+ T cells but was impaired in WSX-1-deficient naive CD4+ T cells. Additionally, IL-27 stimulation induced T-bet and IL-12Rβ2 expression in wild-type, but not in WSX-1-deficient, CD4+ T cells. Thus, during the initiation of Th1 differentiation, the IL-27/WSX-1 signaling system plays a pivotal role by STAT1-mediated T-bet induction before the IL-12R system.

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation1-3. Raf is activated through both Ras-dependent and Ras-independent mechanisms4-6, but the regulatory mechanisms of Raf activation remain unclear7-9. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified10-13 and characterized as negative regulators of growth-factor-induced ERK activation14-25. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling19,25, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.