研究者詳細 - 吉村　昭彦

論文 - 吉村　昭彦

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Mitogen-activated protein kinase plays an essential role in the erythropoietin-dependent proliferation of CTLL-2 cells

Sakamoto H., Kitamura T., Yoshimura A.

Journal of Biological Chemistry 275 （ 46 ） 35857 - 35862 2000年11月

ISSN 0021-9258

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.

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The JAK-inhibitor, JAB/SOCS-1 selectively inhibits, cytokine-induced, but not v-Src induced JAK-STAT activation

Iwamoto Takashi, Senga Takeshi, Naito Yuko, Matsuda Satoru, Miyake Yozo, Yoshimura Akihiko, Hamaguchi Michinari

Oncogene 19 （ 41 ） 4795 - 4801 2000年09月

ISSN 0950-9232

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.

CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2

Sasaki Atsuo, Yasukawa Hideo, Shouda Takanori, Kitamura Toshio, Dikic Ivan, Yoshimura Akihiko

Journal of Biological Chemistry 275 （ 38 ） 29338 - 29347 2000年09月

ISSN 0021-9258

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.

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Intracellular retention and degradation of the epidermal growth factor receptor, Two distinct processes mediated by benzoquinone ansamycins

Supino-Rosin Lia, Yoshimura Akihiko, Yarden Yossef, Elazar Zvulun, Neumann Drorit

Journal of Biological Chemistry 275 （ 29 ） 21850 - 21855 2000年07月

ISSN 0021-9258

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.

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Role of the JAK/STAT pathway in rat carotid artery remodeling after vascular injury

Seki Yukihiko, Kai Hisashi, Shibata Rei, Nagata Tsuyoshi, Yasukawa Hideo, Yoshimura Akihiko, Imaizumi Tsutomu

Circulation Research 87 （ 1 ） 12 - 18 2000年07月

ISSN 0009-7330

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.

SOCS-1 can suppress CD3ζ- and Syk-mediated NF-AT activation in a non-lymphoid cell line

Matsuda Tadashi, Yamamoto Tetsuya, Kishi Hiroyuki, Yoshimura Akihiko, Muraguchi Atsushi

FEBS Letters 472 （ 2-3 ） 235 - 240 2000年04月

ISSN 0014-5793

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.

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Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

Masuhara Masaaki, Nagao Kenji, Nishikawa Mitsuo, Sasaki Mika, Yoshimura Akihiko, Osawa Masatake

Biochemical and Biophysical Research Communications 268 （ 3 ） 697 - 703 2000年02月

ISSN 0006-291X

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.

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The Jak-Stat pathway in normal and perturbed hematopoiesis

Ward Alister C., Touw Ivo, Yoshimura Akihiko

Blood 95 （ 1 ） 19 - 29 2000年01月

ISSN 0006-4971

Signal transduction of cytokine receptors and its regulation

Yoshimura A.

Seikagaku 72 （ 2 ） 75 - 90 2000年

ISSN 0037-1017

IL-5-induced JAB-JAK2 interaction

Zahn Stefan, Godillot Paul, Yoshimura Akihiko, Chaiken Irwin

Cytokine 12 （ 9 ） 1299 - 1306 2000年

ISSN 1043-4666

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.

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Molecular cloning and characterization of interleukin 13 receptor binding protein

Murata Takashi, Kurokawa Kenji, Sasaki Atsuo, Yoshimura Akihiko, Puri Raj K.

Blood 96 （ 11 PART II ） 2000年

ISSN 0006-4971

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.

Negative regulation of cytokine signaling pathways

Yasukawa Hideo, Sasaki Atsuo, Yoshimura Akihiko

Annual Review of Immunology 18 143 - 164 2000年

ISSN 0732-0582

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.

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The janus kinase inhibitor, JAB/SOCS-1, is an interferon-γ inducible gene and determines the sensitivity to interferons

Sakamoto Hiroshi, Kinjyo Ichiko, Yoshimura Akihiko

Leukemia and Lymphoma 38 （ 1-2 ） 49 - 58 2000年

ISSN 1042-8194

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.

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吉村 昭彦

論文 - 吉村 昭彦

吉村　昭彦

論文 - 吉村　昭彦