Language：English   Publishing type：Research paper (scientific journal)  

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome-independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1-interacting E3 ligase using a cell-free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C-terminal region. DTX2-induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1-induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth-inhibitory effect in RUNX1-dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome-independent manner.

DOI： 10.1111/febs.16914

PubMed

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

We established orthogonal deubiquitination, which enables us to selectively observe a single type of deubiquitinating enzyme activity in living cells.

DOI： 10.1039/d3cb00095h

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1^−/−-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

DOI： 10.1038/s41419-022-05145-5

researchmap

Other Link： https://www.nature.com/articles/s41419-022-05145-5

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2021.12.109

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZJas < MED25CMIDM < JAZCMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

DOI： 10.1016/j.jbc.2021.101504

PubMed

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of Myosin light-chain kinase 4 (Mylk4) as a target of AR in skeletal muscles. Mylk4 knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.

DOI： 10.1016/j.isci.2021.102303

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本薬学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

DOI： 10.15252/embj.2020105375

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

DOI： 10.1038/s42003-020-01603-y

PubMed

researchmap

Language：English  

DOI： 10.1016/j.bbrc.2020.12.045

PubMed

researchmap

Publishing type：Research paper (international conference proceedings)  

Cell-free protein production technology can easily produce recombinant proteins from cDNA temples, because it is synthesized in a tube without cell culture. We developed a wheat cell-free protein production system from washed wheat embryos. Since our cell-free system is based on eukaryotic translational machinery, it is very suitable for synthesis of eukaryotic proteins such as human protein. Using this system, recently we made a protein array technology consisting of proteins synthesized in 384-well formatted plates, and then constructed human protein array consisting of more than 20,000 recombinant human proteins (20K-HuPA). In addition, combinations with AlphaScreen or magnetic plate technology promotes development of a new high-throughput and high-sensitive approach for identification of protein–protein or protein–antibody interaction. Herein, we demonstrate the results of protein interactomes and antibody validation by using protein array.

DOI： 10.1007/978-981-16-4866-3_18

Scopus

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/tpj.14955

researchmap

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

DOI： 10.3390/biomedicines8060152

researchmap

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-κB activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-κB signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-κB signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs.

DOI： 10.1016/j.bbrc.2019.12.049

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.

DOI： 10.1039/c9pp00329k

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

The transcription factor GATA3 is a master regulator that modulates T helper 2 (Th2) cell differentiation and induces expression of Th2 cytokines, such as IL-4, IL-5, and IL-13. Th2 cytokines are involved in the protective immune response against foreign pathogens, such as parasites. However, excessive production of Th2 cytokines results in type-2 allergic inflammation. Therefore, the application of a GATA3 inhibitor provides a new therapeutic strategy to regulate Th2 cytokine production. Here, we established a novel high-throughput screening system for an inhibitor of a DNA-binding protein, such as a transcription factor, and identified pyrrothiogatain as a novel inhibitor of GATA3 DNA-binding activity. Pyrrothiogatain inhibited the DNA-binding activity of GATA3 and other members of the GATA family. Pyrrothiogatain also inhibited the interaction between GATA3 and SOX4, suggesting that it interacts with the DNA-binding region of GATA3. Furthermore, pyrrothiogatain significantly suppressed Th2 cell differentiation, without impairing Th1 cell differentiation, and inhibited the expression and production of Th2 cytokines. Our results suggest that pyrrothiogatain regulates the differentiation and function of Th2 cells via inhibition of GATA3 DNA binding activity, which demonstrates the efficiency of our drug screening system for the development of novel small compounds that inhibit the DNA-binding activity of transcription factors.

DOI： 10.1038/s41598-019-53856-1

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

DOI： 10.1074/jbc.RA119.010119

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562 cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562 cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.

DOI： 10.1016/j.bbrc.2018.10.006

PubMed

researchmap

Language：English   Publisher：(一社)日本血液学会-東京事務局  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N′-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

DOI： 10.1016/j.bbrc.2017.12.142

Scopus

PubMed

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1-2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

DOI： 10.1038/s41598-017-12497-y

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia.

DOI： 10.1074/jbc.M117.785675

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4(+) T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-kappa B,which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the I kappa B kinase (IKK) complex, which is a critical step in NF-kappa B activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63-and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation- mediated IKK activation.

DOI： 10.1371/journal.ppat.1006162

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2016.08.010

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Myogenic regulatory factors of the MyoD family have the ability to reprogram differentiated cells toward a myogenic fate. In this study, we demonstrate that Six1 or Six4 are required for the reprogramming by MyoD of mouse embryonic fibroblasts (MEFs). Using microarray experiments, we found 761 genes under the control of both Six and MyoD. Using MyoD ChIPseq data and a genome-wide search for Six1/4 MEF3 binding sites, we found significant co-localization of binding sites for MyoD and Six proteins on over a thousand mouse genomic DNA regions. The combination of both datasets yielded 82 genes which are synergistically activated by Six and MyoD, with 96 associated MyoD+MEF3 putative cis-regulatory modules (CRMs). Fourteen out of 19 of the CRMs that we tested demonstrated in Luciferase assays a synergistic action also observed for their cognate gene. We searched putative binding sites on these CRMs using available databases and de novo search of conserved motifs and demonstrated that the Six/MyoD synergistic activation takes place in a feedforward way. It involves the recruitment of these two families of transcription factors to their targets, together with partner transcription factors, encoded by genes that are themselves activated by Six and MyoD, including Mef2, Pbx-Meis and EBF.

DOI： 10.1093/nar/gkw512

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-kappa B suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-kappa B suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-alpha-mediated NF-kappa B activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-alpha-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-kappa B are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-kappa B activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS.

DOI： 10.1038/ncomms12547

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

The plant cell wall, the most external layer of the plant surface, is the site where most pathogenic fungi first make contact with host cells. A plant–fungus interaction therefore commences at the interface between the plant and the spore. Our current research focusing on the plant cell wall has discovered an extracellular ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase/apyrase
EC3.6.1.15) as a key player in plant defense before the onset of PTI (PAMP-triggered immunity). This review focuses on our recent findings, especially the role of the plant cell wall in the extracellular defense against fungi as well as fungal strategies resulting in successful infection.

DOI： 10.1016/j.pmpp.2016.02.006

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the "AGIA" tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 x 10(-9) M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.

DOI： 10.1371/journal.pone.0156716

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

DOI： 10.1371/journal.pone.0156718

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purificationmass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

DOI： 10.1371/journal.ppat.1005357

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which similar to 470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, connot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised.
Results: Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time.
Conclusion: The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

DOI： 10.1186/s12870-015-0660-9

Web of Science

PubMed

researchmap

Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

Post-translational modifications have crucial roles in the regulation of many physiological processes such as development, differentiation, and response to extracellular signals. Currently, protein phosphorylation is considered as an important trigger for many signal transductions. Protein ubiquitination initially was thought to be a tag for protein degradation
however, recent studies demonstrated that protein ubiquitination also functions as a key regulator of signal transductions. In particular, proteomics analysis using high-sensitivity mass spectrometry provides a large number of modification sites for phosphorylation or ubiquitination of protein in the cells. Although the information from these modifications allows us to image the regulatory mechanism of the protein, it is difficult to know the protein kinase or ubiquitin ligase responsible for these modifications. Recently, we developed a protein array technology based on a wheat germ cell-free protein production system for biochemical analysis. Here, we introduce the procedure of the protein array construction and novel methods using the protein array to identify the responsible enzymes for phosphorylation or ubiquitination of target protein.

DOI： 10.1007/978-4-431-55561-2_4

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multi-step phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. The receiver domain therefore plays a crucial role in controlling the phosphorelay to the response regulator. Furthermore, our in vitro assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro.

DOI： 10.1371/journal.pone.0132598

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The type I interferon system is essential for antiviral immune response and is a primary target of viral immune evasion strategies. Here, we show that virus infection induces the expression of MAPK phosphatase 5 (MKP5), a dual-specificity phosphatase (DUSP), in host cells. Mice deficient in MKP5 were resistant to H1N1 influenza infection, which is associated with increased IRF3 activation and type I interferon expression in comparison with WT mice. Increased type I interferon responses were also observed in MKP5-deficient cells and animals upon other RNA virus infection, including vesicular stomatitis virus and sendai virus. These observations were attributed to the ability of MKP5 to interact with and dephosphorylate IRF3. Our study reveals a critical function of a DUSP in negative regulation of IRF3 activity and demonstrates a mechanism by which influenza and other RNA viruses inhibit type I interferon response in the host through MKP5.

DOI： 10.1016/j.celrep.2015.02.030

Web of Science

PubMed

researchmap

DOI： 10.1371/journal.pone.0132598

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors.

DOI： 10.1074/jbc.M114.561662

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-alpha (CKI-alpha) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-alpha-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-alpha depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-alpha-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle.

DOI： 10.1128/JVI.03170-13

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Retinoblastoma protein (pRB) controls cell cycle progression and cell cycle exit through interactions with cellular proteins. Many pRB-binding proteins, which function in gene transcription or modulation of chromatin structure, harbor LXCXE motifs in their binding domain for pRB. In this study, we found that nuclear mitotic apparatus protein (NuMA), a mitotic spindle organizer, interacts with pRB through LSCEE sequences located in its C-terminal region. siRNA-mediated down-regulation of pRB caused aberrant distribution of NuMA and alignment of spindle microtubules in mitotic cells. Abnormal organization of spindle microtubules was also accompanied by misalignment of an over-expressed NuMA mutant (mut-NuMA) with a defect in pRB binding caused by an LSGEK mutation. The mut-NuMA-over-expressing cells showed lower potency for survival than wild-type NuMA (wt-NuMA)-over-expressing cells during 2weeks of culture. Interestingly, knockdown of pRB reduced the population of wt-NuMA-over-expressing cells to the same level as mut-NuMA cells after 2weeks. Taken together, pRB may have a novel function in regulating the mitotic function of NuMA and spindle organization, which are required for proper cell cycle progression.

DOI： 10.1111/gtc.12119

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Landes Bioscience  

Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes the plant cell death by the inhibiting glutamine synthetase, which leads to an abnormal accumulation of ammonium ions. To better understand the molecular mechanisms involved in TβLinduced cell death and necrotic wildfire lesions, we focused on adenylyl cyclase in Nicotiana benthamiana. We isolated the gene designated as NbAC (Nicotiana benthamiana adenylyl cyclase). Recombinant NbAC protein showed adenylyl cyclase activity in vitro. TβL-induced necrotic lesions were significantly suppressed in NbAC-silenced leaves compared with control plant leaves. However, the amount of ammonium ions was scarcely affected by NbAC-silencing. Furthermore, the silencing of NbAC also suppressed l-methionine sulfoximine-induced cell death without any changes in the amount of ammonium accumulated. When inoculated directly with P. syringae pv tabaci, NbAC-silenced plants showed reduced symptoms. These results suggest that NbAC might play an essential role in intracellular signal transduction during TβL-induced cell death and necrotic wildfire disease development. © 2014 Landes Bioscience.

DOI： 10.4161/psb.27420

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

DOI： 10.1371/journal.pone.0086709

Web of Science

PubMed

researchmap

Language：English   Publisher：(一社)日本エイズ学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Nicotiana benthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive Delta lpp1 Delta dpp1 Delta pah1 mutant yeast. Recombinant DS1 protein showed Mg2+-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

DOI： 10.1371/journal.pone.0075124

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. (c) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.antiviral.2012.09.023

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities.
Structured summary of protein interactions:
PTP1 dephosphorylates MBP by phosphatase assay (View interaction).
AtPP2C dephosphorylates MBP by phosphatase assay (View interaction).
POLTE dephosphorylates MBP by phosphatase assay (View interaction).
TOPP8 dephosphorylates MBP by phosphatase assay (View interaction).
HAB1 dephosphorylates MBP by phosphatase assay (View interaction).
ABI2 dephosphorylates MBP by phosphatase assay (View interaction).
At1g34750 dephosphorylates MBP by phosphatase assay (View interaction).
At1g43900 dephosphorylates MBP by phosphatase assay (View interaction).
At3g15260 dephosphorylates MBP by phosphatase assay (View interaction).
At5g53140 dephosphorylates MBP by phosphatase assay (View interaction).
At1g18030 dephosphorylates MBP by phosphatase assay (View interaction).
At3g06270 dephosphorylates MBP by phosphatase assay (View interaction).
At2g25070 dephosphorylates MBP by phosphatase assay (View interaction).
At3g02750 dephosphorylates MBP by phosphatase assay (View interaction).
At5g10740 dephosphorylates MBP by phosphatase assay (View interaction).
at4g26080 dephosphorylates MBP by phosphatase assay (View interaction).
At4g28400 dephosphorylates MBP by phosphatase assay (View interaction).
At5g06750 dephosphorylates MBP by phosphatase assay (View interaction).
At4g31860 dephosphorylates MBP by phosphatase assay (View interaction).
At3g17250 dephosphorylates MBP by phosphatase assay (View interaction).
At4g38520 dephosphorylates MBP by phosphatase assay (View interaction).
At3g05640 dephosphorylates MBP by phosphatase assay (View interaction).
At5g66080 dephosphorylates MBP by phosphatase assay (View interaction).
At1g79630 dephosphorylates MBP by phosphatase assay (View interaction).
At2g30170 dephosphorylates MBP by phosphatase assay (View interaction).
At5g24940 dephosphorylates MBP by phosphatase assay (View interaction). (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2012.08.014

Web of Science

PubMed

researchmap

Language：English   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses. (C) 2012 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.jplph.2012.04.002

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Protein phosphorylation is one of the main process in the signal transduction pathway. In recent years, there has been increasing attention to plant phosphorylation signaling and many laboratories are trying to elucidate pathways using various approaches. Although more than 1000 protein kinase (PK) genes have been annotated in the Arabidopsis genome, biochemical characterization of those PKs is limited. In this work, we demonstrate high-throughput profiling of serine/threonine autophosphorylation activity by a combination of the 759N-terminal biotinylated proteins library, produced using a wheat germ cell-free protein production system, and a commercially available luminescence system. Luminescent analysis revealed that 179 of the 759 PKs had autophosphorylation activity. From these 179 PKs, 67 of the most active PKs were analyzed to determine their function using the PlantP database. This analysis revealed that 35(53%) of the proteins were classified as non-transmembrane protein kinases, and 15(23%) were receptor-like protein kinases. Additionally, PKs from Group 4.4-MAP3K, Group 1.6, Group 4.5-MAPK/CDC/CK2/GSK kinases and Group 1.10-receptor like cytoplasmic kinases contained the highest percentage of autophosphorylated activity. Next, to get a better overview of the annotated 67 PK5, we used the gene ontology annotation search on the TAIR website to classify the 67 PKs into functional category. As a result, some of these PKs may be involved in phospho-signaling pathways such as signal transduction, stress response, and the regulation of cell division. Information from this study may shed light on many unknown plant PICs. This study will be a basis for understanding the function of PKs in phosphorylation network for future research. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2011.02.029

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Plant Ca(2+) signals are involved in a wide array of intracellular signalling pathways after pest invasion. Ca(2+)-binding sensory proteins such as Ca(2+)-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca(2+) influx after insect herbivory. However, until now this prediction was not testable.
Results: To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca(2+) levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca(2+). CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca(2+). Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.
Conclusions: These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

DOI： 10.1186/1471-2229-10-97

Web of Science

PubMed

researchmap

Publisher：The Japanese Society of Plant Physiologists  

Calcium-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. In vitro kinase assays of the CPK proteins with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including HsfB2a). In vivo agroinflitaration assays showed that CPK-derived phosphorylation of HsfB2a promotes PDF1.2 transcriptional activation in defense response. Furthermore, both CPKs are involved in the transcript level of heat shock protein (HSP) genes, which resulted in impairment of basal thermotolerance in cpk3 and cpk13 mutants. However, HsfB2a played a role in HSP transcripts as suppressor. These results reveal an intricate array of CPK-associated signal transduction networks that are in part common, but by distinct mechanisms, between plant-insect interactions and heat-shock signal transduction.

DOI： 10.14841/jspp.2010.0.0312.0

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We created a protein library consisting of 647 Arabidopsis transcription factors (TFs) using a wheat cell-free system. The quality of proteins in the library was checked by binding assay of bZIP family proteins. Screening of TFs binding to 5'-regulatory regions of FLC and LFY was conducted using the library, and MYB67 and GBF1 were found to be binding factors.

DOI： 10.1271/bbb.90026

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis.

DOI： 10.1093/jxb/erp134

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.
Results: Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG- tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.
Conclusion: In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.

DOI： 10.1186/1471-2229-9-39

Web of Science

PubMed

researchmap

Publisher：The Japanese Society of Plant Physiologists  

To avoid the generation of reactive oxygen species, makeup of PS II and PS I must be synchronized. We have found that a major sigma factor, SIG1, in RNA polymerase is phosphorylated in dim light to suppress the expression of photosynthesis genes, whereas SIG1 is dephosphorylated under irradiance to release the suppression.<br><br> We have employed a wheat germ cell-free protein production system in combination with AlphaScreen (PerkinElmer) to detect protein-protein interaction <I>in vitro</I> to search protein kinases responsible for phosphorylation of SIG1. SIG1 and approx. 800 protein kinases have been synthesized <I>in vitro</I> using RIKEN <I>Arabidopsis</I> Full-Length (RAFL) clones and subjected to the screening. We have obtained 49 candidate protein kinases. A phosphorylation assay has shown an activity to phosphorylate the Thr-170 of SIG1 in the wheat germ extracts.

DOI： 10.14841/jspp.2009.0.0452.0

researchmap

Publisher：The Japanese Society of Plant Physiologists  

Protein ubiquitination is implicated in several critical cellular processes. Ubiquitination is mediated by the sequential action of at least three enzymes, the E1, E2 and E3 proteins. Although Arabidopsis genome research estimates over 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrated a novel method for high-throughput in vitro ubiquitination analysis based on wheat cell-free protein synthesis and luminescent detection. The recombinant proteins without purification were used for the assay, and luminescent analysis showed the ubiquitin-conjugation of 29 E2s and a HECT-type E3. Furthermore, this analysis also detected the polyubiquitination of a HECT- and RING-type E3s. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. Based on this study, we are developing the convenient and versatile method to elucidate plant ubiquitination pathway.

DOI： 10.14841/jspp.2009.0.0532.0

researchmap

Publisher：The Japanese Society of Plant Physiologists  

Ca2+ signals have evolved a wide spectrum of strategies as intra/intercellular signals. Even though Ca2+-binding sensory proteins like Ca2+-dependent protein kinase (CDPK) are expected to act as intracellular protein mediators that govern gene regulation networks in response to Ca2+ influx after herbivory, little is known about the mechanisms. To investigate whether CDPKs play certain roles in an herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CDPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. After herbivory, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. in vitro Kinase assays of the CDPK proteins with defense-related transcription factors (TFs) demonstrated that CPK3 phosphorylates TFs and ATL2 as substrate targets. The results presented show that CDPKs are involved in controlling the herbivore-induced expression of plant defensin gene, and that they exert such control by interacting with transcription factors.

DOI： 10.14841/jspp.2009.0.0088.0

researchmap

Language：English   Publisher：OXFORD UNIV PRESS  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Apyrases (E.C.3.6.1.5
NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-006-0279-7

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-005-0267-3

Scopus

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Authorship：Lead author,　Corresponding author   Language：Japanese  

researchmap

Authorship：Lead author,　Corresponding author   Language：Japanese  

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：大阪市医学会  

J-GLOBAL

researchmap

Language：English  

DOI： 10.1016/j.bbamcr.2016.08.010

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.cyto.2014.07.219

Web of Science

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publisher：(一社)日本細胞生物学会  

researchmap

Language：Japanese   Publisher：(有)科学評論社  

網羅的手法を用いて、C型肝炎ウイルス(HCV)の非構造蛋白であるNS5A蛋白のリン酸化に関与する新規プロテインキナーゼの同定を行った。方法として、NS5A蛋白をコムギ胚芽無細胞転写・翻訳系で合成した。また、404種類のヒトプロテインキナーゼを包括するcDNAライブラリーから同様の方法でプロテインキナーゼを取得した。NS5A蛋白とプロテインキナーゼの相互作用をAlpha Screen法で解析し、79種類のセリン/スレオニンプロテインキナーゼを同定した。次に、NS5A蛋白に対するリン酸化能を評価するため、79種類のセリン/スレオニンプロテインキナーゼに対しin vitroリン酸化アッセイを行い、9種類にNS5A蛋白に対し強いリン酸化活性を認めた。更に、HCVゲノム複製能をサブゲノミックレプリコンRNAを用いて、また、粒子形成能を全長HCV RNAもしくは感染性ウイルス粒子を用いて解析し、感染性HCV産生を制御する2種類の新規プロテインキナーゼを同定した。

researchmap

Language：Japanese   Publisher：(一社)日本肝臓学会  

researchmap

Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Ca^<2+> signals have evolved a wide spectrum of strategies as intra/intercellular signals. Even though Ca^<2+>-binding sensory proteins like Ca^<2+>-dependent protein kinase (CDPK) are expected to act as intracellular protein mediators that govern gene regulation networks in response to Ca^<2+> influx after herbivory, little is known about the mechanisms. To investigate whether CDPKs play certain roles in an herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CDPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. After herbivory, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. These CDPKs appear to be located mainly in the nucleus/cytosol, and in vitro kinase assays of the CDPK proteins with 87 defense-related transcription factors (TFs), synthesized from the wheat germ cell-free system, demonstrated that CPK3 phosphorylates TFs and ATL2 (a member of the RING-H2 zinc finger proteins that may function as E3 ubiquitin ligase) as substrate targets. Rather, while CPK13 does not phosphorylate any of them, this kinase is predicted to be involved in the green leaf volatile-signaling pathway. The results presented show that CPK3 and CPK13 are involved in controlling the herbivore-induced expression of plant defensin gene, and that they exert such control through distinct mechanisms by directly interacting with transcription factors or volatile-mediated gene regulation.

CiNii Books

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

CiNii Books

researchmap

Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

CiNii Books

researchmap

Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

CiNii Books

researchmap

Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

CiNii Books

researchmap

Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

CiNii Books

researchmap

Event date： 2022.11

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.11

Presentation type：Poster presentation  

researchmap

Event date： 2022.11

Presentation type：Poster presentation  

researchmap

Event date： 2022.11

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.11

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.11

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Event date： 2022.10

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.10

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.10

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.9

Presentation type：Poster presentation  

researchmap

Event date： 2022.9

Presentation type：Poster presentation  

researchmap

Event date： 2022.8

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Event date： 2022.5

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.5

Presentation type：Oral presentation (general)  

researchmap

Event date： 2020.12

Language：English  

researchmap

Event date： 2020.11

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Event date： 2020.9

Language：English   Presentation type：Oral presentation (invited, special)  

researchmap

Event date： 2019.12

Language：English   Presentation type：Oral presentation (invited, special)  

researchmap

Event date： 2019.12

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2019.11

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Event date： 2019.10

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2019.10

Language：English   Presentation type：Poster presentation  

researchmap

Event date： 2019.9

Language：English   Presentation type：Poster presentation  

researchmap

Event date： 2019.8

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2018.1

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Event date： 2017.12

Language：Japanese   Presentation type：Oral presentation (invited, special)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：パシフィコ横浜  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (invited, special)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Oral presentation (invited, special)  

researchmap

Language：English   Presentation type：Oral presentation (invited, special)  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：名古屋大学・創薬科学研究館  

researchmap

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：大阪大学微生物病研究所  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪大学医学部 銀杏会館  

researchmap

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：神奈川県 湯河原温泉 ホテルあかね  

researchmap

Language：English   Presentation type：Poster presentation  

Venue：Beaver Run Resort, Breckenridge, Colorado, USA  

researchmap

Presentation type：Oral presentation (general)  

Venue：パシフィコ横浜  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：パシフィコ横浜  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：国立京都国際会館  

researchmap

Language：English   Presentation type：Oral presentation (general)  

Venue：Whistler Conference Centre, Whistler, British Columbia, Canada  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸ポートアイランド  

researchmap

Language：English   Presentation type：Oral presentation (general)  

Venue：福岡国際会議場  

researchmap

Language：English   Presentation type：Poster presentation  

Venue：Strasbourg, France  

researchmap

Language：Japanese   Presentation type：Oral presentation (invited, special)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長野県茅野市  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：グランキューブ大阪（大阪国際会議場）  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋大学  

researchmap

Language：Japanese   Presentation type：Poster presentation  

Venue：神戸ポートアイランド  

researchmap

Language：Japanese   Presentation type：Poster presentation  

Venue：神戸ポートアイランド  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸国際会議場  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：群馬県 伊香保温泉  

researchmap