Wendell Lim(著), Bruce Mayer(著), Tony Pawson(著), 西田 栄介(監訳), 小迫 英尊 : 細胞のシグナル伝達, 第9章, メディカル・サイエンス・インターナショナル, 東京, 2016年5月.
2.
小迫 英尊, 後藤 由季子 : タンパク質のリン酸化, 朝倉書店, 2008年7月.
3.
Kaori Matsuzawa, Hidetaka Kosako, Ichiro Azuma, Naoyuki Inagaki and Masaki Inagaki : Possible regulation of intermediate filament proteins by Rho-binding kinases., Plenum Press, 1998.
学術論文(審査論文):
1.
Kazuki Okuyama, Aneela Nomura, Kohei Nishino, Hirokazu Tanaka, Christelle Harly, Risa Chihara, Yasuyo Harada, Sawako Muroi, Masato Kubo, Hidetaka Kosako and Ichiro Taniuchi : The Majority of the Serine/Threonine Phosphorylation Sites in Bcl11b Protein Are Dispensable for the Differentiation of T Cells., The Journal of Immunology, Vol.210, No.11, 1728-1739, 2023.
(要約)
Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.
Ryuto Tsuchiya, Yuki Yoshimatsu, Rei Noguchi, Yooksil Sin, Takuya Ono, Taro Akiyama, Hidetaka Kosako, Akihiko Yoshida, Seiji Ohtori, Akira Kawai and Tadashi Kondo : Integrating analysis of proteome profile and drug screening identifies therapeutic potential of MET pathway for the treatment of malignant peripheral nerve sheath tumor., Expert Review of Proteomics, Vol.20, No.4-6, 109-119, 2023.
(要約)
We successfully identified novel therapeutic candidates for the treatment of MPNST, namely crizotinib and foretinib, which target the MET pathway. We hope that these candidate drugs will contribute to the treatment of MPNST.
Shiori Akabane, Kiyona Watanabe, Hidetaka Kosako, Shun-Ichi Yamashita, Kohei Nishino, Masahiro Kato, Shiori Sekine, Tomotake Kanki, Noriyuki Matsuda, Toshiya Endo and Toshihiko Oka : TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria., Cell Reports, Vol.42, No.5, 112454, 2023.
(要約)
PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.
Ikuko Maejima, Taichi Hara, Satoshi Tsukamoto, Hiroyuki Koizumi, Takeshi Kawauchi, Tomoko Akuzawa, Rika Hirai, Hisae Kobayashi, Inoya Isobe, Kazuo Emoto, Hidetaka Kosako and Ken Sato : RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution., Communications Biology, Vol.6, No.1, 2023.
(要約)
RAB35 is a multifunctional small GTPase that regulates endocytic recycling, cytoskeletal rearrangement, and cytokinesis. However, its physiological functions in mammalian development remain unclear. Here, we generated Rab35-knockout mice and found that RAB35 is essential for early embryogenesis. Interestingly, brain-specific Rab35-knockout mice displayed severe defects in hippocampal lamination owing to impaired distribution of pyramidal neurons, although defects in cerebral cortex formation were not evident. In addition, Rab35-knockout mice exhibited defects in spatial memory and anxiety-related behaviors. Quantitative proteomics indicated that the loss of RAB35 significantly affected the levels of other RAB proteins associated with endocytic trafficking, as well as some neural cell adhesion molecules, such as contactin-2. Collectively, our findings revealed that RAB35 is required for precise neuronal distribution in the developing hippocampus by regulating the expression of cell adhesion molecules, thereby influencing spatial memory.
M Ahmed Refaat, Mikiyo Nakata, Afzal Husain, Hidetaka Kosako, Tasuku Honjo and A Nasim Begum : HNRNPU facilitates antibody class-switch recombination through C-NHEJ promotion and R-loop suppression., Cell Reports, Vol.42, No.3, 2023.
(要約)
B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S) regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability.
(キーワード)
DNA / DNA Breaks, Double-Stranded / DNA End-Joining Repair / DNA, Single-Stranded / DNA-Binding Proteins / Immunoglobulin Class Switching / Immunoglobulin Isotypes / R-Loop Structures / RNA / Heterogeneous-Nuclear Ribonucleoprotein U
Yuma Horii, Shoichi Matsuda, Chikashi Toyota, Takumi Morinaga, Takeo Nakaya, Soken Tsuchiya, Masaki Ohmuraya, Takanori Hironaka, Ryo Yoshiki, Kotaro Kasai, Yuto Yamauchi, Noburo Takizawa, Akiomi Nagasaka, Akira Tanaka, Hidetaka Kosako and Michio Nakaya : VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid-liquid phase separation., Nature Communications, Vol.14, No.1, 2023.
(要約)
Myofibroblasts cause tissue fibrosis by producing extracellular matrix proteins, such as collagens. Humoral factors like TGF-β, and matrix stiffness are important for collagen production by myofibroblasts. However, the molecular mechanisms regulating their ability to produce collagen remain poorly characterised. Here, we show that vestigial-like family member 3 (VGLL3) is specifically expressed in myofibroblasts from mouse and human fibrotic hearts and promotes collagen production. Further, substrate stiffness triggers VGLL3 translocation into the nucleus through the integrin β1-Rho-actin pathway. In the nucleus, VGLL3 undergoes liquid-liquid phase separation via its low-complexity domain and is incorporated into non-paraspeckle NONO condensates containing EWS RNA-binding protein 1 (EWSR1). VGLL3 binds EWSR1 and suppresses miR-29b, which targets collagen mRNA. Consistently, cardiac fibrosis after myocardial infarction is significantly attenuated in Vgll3-deficient mice, with increased miR-29b expression. Overall, our results reveal an unrecognised VGLL3-mediated pathway that controls myofibroblasts' collagen production, representing a novel therapeutic target for tissue fibrosis.
Kou Motani, Noriko Saito-Tarashima, Kohei Nishino, Shunya Yamauchi, Noriaki Minakawa and Hidetaka Kosako : The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER, Cell Reports, Vol.41, No.12, 111868, 2022.
(要約)
STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis-SATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING.
(キーワード)
Ligands / Proteomics / Membrane Proteins / Endoplasmic Reticulum / Golgi Apparatus / Interferon Type I / Protein Transport
Susumu Katsuma, Kanako Hirota, Noriko Matsuda-Imai, Takahiro Fukui, Tomohiro Muro, Kohei Nishino, Hidetaka Kosako, Keisuke Shoji, Hideki Takanashi, Takeshi Fujii, Shin-Ichi Arimura and Takashi Kiuchi : A Wolbachia factor for male killing in lepidopteran insects., Nature Communications, Vol.13, No.1, 6764, 2022.
(要約)
Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression of Oscar inhibits Masc-induced masculinization and leads to male killing in two lepidopteran insects, O. furnacalis and the silkworm Bombyx mori. Our study identifies a mechanism by which Wolbachia induce male killing of host progeny.
Kohei Nishino, Harunori Yoshikawa, Kou Motani and Hidetaka Kosako : Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics., Journal of Proteome Research, 2022.
(要約)
Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.
Daisuke Oikawa, Min Gi, Hidetaka Kosako, Kouhei Shimizu, Hirotaka Takahashi, Masayuki Shiota, Shuhei Hosomi, Keidai Komakura, Hideki Wanibuchi, Daisuke Tsuruta, Tatsuya Sawasaki and Fuminori Tokunaga : OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways., Cell Death & Disease, Vol.13, No.8, 694, 2022.
(要約)
-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.
(キーワード)
Animals / Cell Death / Deubiquitinating Enzymes / Humans / Kelch-Like ECH-Associated Protein 1 / Mice / NF-E2-Related Factor 2 / NF-kappa B / Reactive Oxygen Species / Ubiquitin / Ubiquitin-Specific Proteases / Ubiquitination
Hiroya Yamazaki, Masatoshi Takagi, Hidetaka Kosako, Tatsuya Hirano and H Shige Yoshimura : Cell cycle-specific phase separation regulated by protein charge blockiness., Nature Cell Biology, Vol.24, No.5, 625-632, 2022.
(要約)
Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and increases their propensity for liquid-liquid phase separation (LLPS). A phosphomimetic sequence and the sequences with enhanced charge blockiness underwent strong LLPS in vitro and induced chromosome periphery formation in vivo. Conversely, mitotic hyperphosphorylation of NPM1 diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution. Cell cycle-specific phase separation can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites.
Takumi Maruhashi, Daisuke Sugiura, Il-mi Okazaki, Kenji Shimizu, K Takeo Maeda, Jun Ikubo, Harunori Yoshikawa, Katsumi Maenaka, Naozumi Ishimaru, Hidetaka Kosako, Tatsuya Takemoto and Taku Okazaki : Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity., Immunity, Vol.55, No.5, 912-924.e8, 2022.
(要約)
Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity invitro and invivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced Tcell suppression invitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity invitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.
(キーワード)
Animals / Antigens, CD / Autoimmunity / Histocompatibility Antigens Class II / Ligands / Mice / Mice, Inbred C57BL / Mice, Inbred NOD / Neoplasms / Peptides / T-Lymphocytes
Harunori Yoshikawa, Kohei Nishino and Hidetaka Kosako : Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE., Journal of Proteomics, Vol.258, 2022.
(要約)
The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases. SIGNIFICANCE: Label-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.
(キーワード)
Animals / Chromatography, Liquid / Electrophoresis, Polyacrylamide Gel / Extracellular Signal-Regulated MAP Kinases / Mice / NIH 3T3 Cells / Phosphoproteins / Phosphorylation / Pyridines / Tandem Mass Spectrometry
Ryouhei Shioya, Kohdai Yamada, Kohki Kido, Hirotaka Takahashi, Akira Nozawa, Hidetaka Kosako and Tatsuya Sawasaki : A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID., Biochemical and Biophysical Research Communications, Vol.592, 54-59, 2022.
(要約)
Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.
Masato Miyake, Mitsuaki Sobajima, Kiyoe Kurahashi, Akira Shigenaga, Masaya Denda, Akira Otaka, Tomohide Saio, Naoki Sakane, Hidetaka Kosako and Seiichi Oyadomari : Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells., Cell Chemical Biology, Vol.29, No.6, 996-1009.e9, 2022.
(要約)
Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function.
Satoshi Yamanaka, Yuto Horiuchi, Saya Matsuoka, Kohki Kido, Kohei Nishino, Mayaka Maeno, Norio Shibata, Hidetaka Kosako and Tatsuya Sawasaki : A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues., Nature Communications, Vol.13, No.1, 2022.
(要約)
Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein-responsible for the 8p11 syndrome involved in acute myeloid leukaemia-as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein-protein interactions in cells.
Takaharu Sakuragi, Ryuta Kanai, Akihisa Tsutsumi, Hirotaka Narita, Eriko Onishi, Kohei Nishino, Takuya Miyazaki, Takeshi Baba, Hidetaka Kosako, Atsushi Nakagawa, Masahide Kikkawa, Chikashi Toyoshima and Shigekazu Nagata : The tertiary structure of the human Xkr8-Basigin complex that scrambles phospholipids at plasma membranes., Nature Structural & Molecular Biology, Vol.28, No.10, 825-834, 2021.
(要約)
Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.
ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.
Akihito Morita, Yuhkoh Satouh, Hidetaka Kosako, Hisae Kobayashi, Akira Iwase and Ken Sato : Clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins and preimplantation development., Development, Vol.148, No.14, dev199461, 2021.
(要約)
Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.
Ariel Pradipta, Miwa Sasai, Kou Motani, Su Ji Ma, Youngae Lee, Hidetaka Kosako and Masahiro Yamamoto : killing program requires Irgm2 but not its microbe vacuolar localization., Life Science Alliance, Vol.4, No.7, e202000960, 2021.
Miwa Sasai, Su Ji Ma, Masaaki Okamoto, Kohei Nishino, Hikaru Nagaoka, Eizo Takashima, Ariel Pradipta, Youngae Lee, Hidetaka Kosako, Pann-Ghill Suh and Masahiro Yamamoto : Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells., The Journal of Experimental Medicine, Vol.218, No.7, e20201763, 2021.
(要約)
Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4+ and CD8+ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8+ T cells is qualitatively different from that in CD4+ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8+ T cells, whereas those in CD4+ T cells were intact. PLCβ4-deficient CD8+ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP3 generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8+ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8+ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4+ and CD8+ T cells and selectively promotes CD8+ T cell-dependent adaptive immunity.
Masahiro Maruoka, Panpan Zhang, Hiromi Mori, Eiichi Imanishi, M Daniel Packwood, Hiroshi Harada, Hidetaka Kosako and Jun Suzuki : Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane., Molecular Cell, Vol.81, No.7, 1397-1410.e9, 2021.
(要約)
Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.
Yuka Takehara, Hideki Yashiroda, Yoshitaka Matsuo, Xian Zhao, Akane Kamigaki, Tetsuo Matsuzaki, Hidetaka Kosako, Toshifumi Inada and Shigeo Murata : The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation., iScience, Vol.24, No.3, 102145, 2021.
(要約)
Δ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle.
Waka Kojima, Koji Yamano, Hidetaka Kosako, Kenichiro Imai, Reika Kikuchi, Keiji Tanaka and Noriyuki Matsuda : Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy., Autophagy, Vol.17, No.8, 2011-2036, 2021.
(要約)
phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity.
Koichiro Yamashita, Shigehiko Tamura, Masanori Honsho, Hiroto Yada, Yuichi Yagita, Hidetaka Kosako and Yukio Fujiki : Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery., The Journal of Cell Biology, Vol.219, No.10, e202001003, 2020.
(要約)
Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.
Junji Chida, Hideyuki Hara, Keiji Uchiyama, Etsuhisa Takahashi, Hironori Miyata, Hidetaka Kosako, Yukiko Tomioka, Toshihiro Ito, Hiroyuki Horiuchi, Haruo Matsuda, Hiroshi Kido and Suehiro Sakaguchi : Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice., PLoS Pathogens, Vol.16, No.8, e1008823, 2020.
(要約)
The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.
Kou Motani and Hidetaka Kosako : BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)., The Journal of Biological Chemistry, Vol.295, No.32, 11174-11183, 2020.
(要約)
Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. Global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull-down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only non-biotinylated peptides are identified, which cannot be distinguished from peptides of non-specifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.
Eiichi Hashimoto, Shota Okuno, Shoshiro Hirayama, Yoshiyuki Arata, Tsuyoshi Goto, Hidetaka Kosako, Jun Hamazaki and Shigeo Murata : Enhanced O-GlcNAcylation Mediates Cytoprotection under Proteasome Impairment by Promoting Proteasome Turnover in Cancer Cells., iScience, Vol.23, No.7, 101299, 2020.
(要約)
The proteasome is a therapeutic target in cancer, but resistance to proteasome inhibitors often develops owing to the induction of compensatory pathways. Through a genome-wide siRNA screen combined with RNA sequencing analysis, we identified hexokinase and downstream O-GlcNAcylation as cell survival factors under proteasome impairment. The inhibition of O-GlcNAcylation synergistically induced massive cell death in combination with proteasome inhibition. We further demonstrated that O-GlcNAcylation was indispensable for maintaining proteasome activity by enhancing biogenesis as well as proteasome degradation in a manner independent of Nrf1, a well-known compensatory transcription factor that upregulates proteasome gene expression. Our results identify a pathway that maintains proteasome function under proteasome impairment, providing potential targets for cancer therapy.
Kohki Kido, Satoshi Yamanaka, Shogo Nakano, Kou Motani, Souta Shinohara, Akira Nozawa, Hidetaka Kosako, Sohei Ito and Tatsuya Sawasaki : AirID, a Novel Proximity Biotinylation Enzyme, for Analysis of Protein-Protein Interactions, eLife, Vol.9, No.e54983, 2020.
(要約)
Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
Takumi Koshiba and Hidetaka Kosako : Mass spectrometry-based methods for analyzing the mitochondrial interactome in mammalian cells., The Journal of Biochemistry, Vol.167, No.3, 225-231, 2020.
(要約)
Protein-protein interactions are essential biologic processes that occur at inter- and intra-cellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein-protein interaction networks in living cells. The combination of proximity-dependent labeling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein-protein interactions in mitochondrial subcellular compartments.
Hiroyuki Kondo, Takafumi Matsumura, Mari Kaneko, Kenichi Inoue, Hidetaka Kosako, Masahito Ikawa, Yousuke Takahama and Izumi Ohigashi : PITHD1 is a proteasome-interacting protein essential for male fertilization, The Journal of Biological Chemistry, Vol.295, No.6, 1658-1672, 2020.
(要約)
The proteasome is a protein-degrading molecular complex that is necessary for protein homeostasis and various biological functions, including cell cycle regulation, signal transduction, and immune response. Proteasome activity is finely regulated by a variety of proteasome-interacting molecules. PITHD1 is a recently described molecule that has a domain putatively capable of interacting with the proteasome. However, it is unknown whether PITHD1 can actually bind to proteasomes and what it does Here we report that PITHD1 is detected specifically in the spermatids in the testis and the cortical thymic epithelium in the thymus. Interestingly, PITHD1 associates with immunoproteasomes in the testis, but not with thymoproteasomes in the thymus. Mice deficient in PITHD1 exhibit severe male infertility accompanied with morphological abnormalities and impaired motility of spermatozoa. Furthermore, PITHD1 deficiency reduces proteasome activity in the testis and alters the amount of proteins that are important for fertilization capability by the sperm. However, the PITHD1-deficient mice demonstrate no detectable defects in the thymus, including T cell development. Collectively, our results identify PITHD1 as a proteasome-interacting protein that plays a nonredundant role in the male reproductive system.
Hiroya Yamazaki, Hidetaka Kosako and Shige H Yoshimura : Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1868, No.1, 140295, 2020.
(要約)
Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4100 and > 2200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.
Keisuke Kitakaze, Shusuke Taniuchi, Eri Kawano, Yoshimasa Hamada, Masato Miyake, Miho Oyadomari, Hirotatsu Kojima, Hidetaka Kosako, Tomoko Kuribara, Suguru Yoshida, Takamitsu Hosoya and Seiichi Oyadomari : Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity., eLife, Vol.8, e43302, 2019.
(要約)
]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.
Fumika Koyano, Koji Yamano, Hidetaka Kosako, Yoko Kimura, Mayumi Kimura, Yukiko Fujiki, Keiji Tanaka and Noriyuki Matsuda : Parkin-mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes., EMBO Reports, Vol.20, No.12, e47728, 2019.
(要約)
Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria.
We previously reported the identification of a novel antimitotic agent with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-iodobenzohydrazide (code number NP-10). However, the mechanism(s) underlying the cancer cell-selective inhibition of mitotic progression by NP-10 remains unclear. Here, we identified NP-10-interacting proteins by affinity purification from HeLa cell lysates using NP-10-immobilized beads followed by mass spectrometry. The results showed that several mitosis-associated factors specifically bind to active NP-10, but not to an inactive NP-10 derivative. Among them, NUP155 and importin β may be involved in NP-10-mediated mitotic arrest. Because NP-10 did not show antitumor activity in vivo in a previous study, we synthesized 19 NP-10 derivatives to identify more effective NP-10-related compounds. HMI83-2, an NP-10-related compound with a Cl moiety, inhibited HCT116 cell tumor formation in nude mice without significant loss of body weight, suggesting that HMI83-2 is a promising lead compound for the development of novel antimitotic agents.
Takahiro Yoshinaka, Hidetaka Kosako, Takuma Yoshizumi, Ryo Furukawa, Yu Hirano, Osamu Kuge, Taro Tamada and Takumi Koshiba : Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region, iScience, Vol.27, No.19, 1065-1078, 2019.
(要約)
The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.
Fumika Koyano, Koji Yamano, Hidetaka Kosako, Keiji Tanaka and Noriyuki Matsuda : Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL, The Journal of Biological Chemistry, Vol.294, No.26, 10300-10314, 2019.
(要約)
() and () are causal genes of recessive familial Parkinson's disease. Parkin is a ubiquitin ligase E3 that conjugates ubiquitin to impaired mitochondrial proteins for organelle degradation. PINK1, a Ser/Thr kinase that accumulates only on impaired mitochondria, phosphorylates two authentic substrates, the ubiquitin-like domain of Parkin and ubiquitin. Our group and others have revealed that both the subcellular localization and ligase activity of Parkin are regulated through interactions with phosphorylated ubiquitin. Once PINK1 localizes on impaired mitochondria, PINK1-catalyzed phosphoubiquitin recruits and activates Parkin. Parkin then supplies a ubiquitin chain to PINK1 for phosphorylation. The amplified ubiquitin functions as a signal for the sequestration and degradation of the damaged mitochondria. Although a bewildering variety of Parkin substrates have been reported, the basis for Parkin substrate specificity remains poorly understood. Moreover, the mechanism underlying initial activation and translocation of Parkin onto mitochondria remains unclear, because the presence of ubiquitin on impaired mitochondria is thought to be a prerequisite for the initial PINK1 phosphorylation process. Here, we show that artificial mitochondria-targeted proteins are ubiquitylated by Parkin, suggesting that substrate specificity of Parkin is not determined by its amino acid sequence. Moreover, recruitment and activation of Parkin are delayed following depletion of the mitochondrial E3, MITOL/March5. We propose a model in which the initial step in Parkin recruitment and activation requires protein ubiquitylation by MITOL/March5 with subsequent PINK1-mediated phosphorylation. Because PINK1 and Parkin amplify the ubiquitin signal via a positive feedback loop, the low substrate specificity of Parkin might facilitate this amplification process.
Yuichi Takashi, Hidetaka Kosako, Shun Sawatsubashi, Yuka Kinoshita, Nobuaki Ito, Maria K. Tsoumpra, Masaomi Nangaku, Masahiro Abe, Munehide Matsuhisa, Shigeaki Kato, Toshio Matsumoto and Seiji Fukumoto : Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation, Proceedings of the National Academy of Sciences of the United States of America, Vol.116, No.23, 11418-11427, 2019.
(要約)
Fibroblast growth factor (FGF) 23 produced by bone is a hormone that decreases serum phosphate (Pi). Reflecting its central role in Pi control, serum FGF23 is tightly regulated by serum Pi alterations. FGF23 levels are regulated by the transcriptional event and posttranslational cleavage into inactive fragments before its secretion. For the latter, O-glycosylation of FGF23 by gene product prevents the cleavage, leading to an increase in serum FGF23. However, the molecular basis of Pi sensing in the regulation of serum FGF23 remains elusive. In this study, we showed that high Pi diet enhanced the skeletal expression of , but not , with expected increases in serum FGF23 and Pi in mice. induction by high Pi was further observed in osteoblastic UMR 106 cells, and this was mediated by activation of the extracellular signal-regulated kinase (ERK) pathway. Through proteomic searches for the upstream sensor for high Pi, we identified one subtype of the FGF receptor (FGFR1c), which was phosphorylated by high Pi in the absence of FGFs. The mode of unliganded FGFR activation by high Pi appeared different from that of FGFR bound to a canonical FGFR ligand (FGF2) when phosphorylation of the FGFR substrate 2α and ERK was monitored. Finally, we showed that an FGFR inhibitor and conditional deletion of in osteoblasts/osteocytes abrogated high Pi diet-induced increases in serum FGF23 and femoral expression in mice. Thus, these findings uncover an unrecognized facet of unliganded FGFR function and illustrate a Pi-sensing pathway involved in regulation of FGF23 production.
Takaharu Sakuragi, Hidetaka Kosako and Shigekazu Nagata : Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure, Proceedings of the National Academy of Sciences of the United States of America, Vol.116, No.8, 2907-2912, 2019.
(要約)
The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the flippase activity normally counteracted Xkr8's ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and flippase down-regulation.
Kou Motani and Hidetaka Kosako : Phosphoproteomic identification and functional characterization of protein kinase substrates by 2D-DIGE and Phos-tag PAGE, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1867, 57-61, 2019.
(要約)
Protein phosphorylation is one of the most common post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by more than 500 genes in higher eukaryotes and play central roles in various cellular signaling pathways. Consequently, genetic abnormalities of protein kinases have been implicated in many diseases. To fully understand the complex phosphorylation-mediated signaling networks, it is important to globally identify and functionally characterize in vivo substrates of individual protein kinases. Advances in electrophoresis-based phosphoproteomic technologies such as two-dimensional difference gel electrophoresis (2D-DIGE) following immobilized metal affinity chromatography (IMAC) and phosphate-affinity Phos-tag PAGE have enabled efficient and detailed analysis of protein kinase substrates. Here, we describe physiological functions of the newly identified substrates of several disease-related protein kinases including ERK, PKD and PINK1.
Takeshi Terabayashi, Katsuhiro Hanada, Kou Motani, Hidetaka Kosako, Mami Yamaoka, Toshihide Kimura and Toshimasa Ishizaki : Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment, Genes to Cells, Vol.23, No.6, 466-479, 2018.
(要約)
During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non-neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin-332 (LN-332) as a principle paracrine factor in conditioned medium from mammary epithelium-derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA-MB-231 cells. Then, we carried out a morphology-based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA-MB-231 cells that were induced by conditioned medium from MCF10A cells and LN-332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium-treated MDA-MB-231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.
Kou Motani and Hidetaka Kosako : Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune semaphorin SEMA4D., The Journal of Biological Chemistry, Vol.293, No.20, 7717-7726, 2018.
(要約)
Stimulator of interferon genes (STING) is an endoplasmic reticulum-resident membrane protein that mediates cytosolic pathogen DNA-induced innate immunity and inflammatory responses in host defenses. STING is activated by cyclic di-nucleotides and is then translocated to the Golgi apparatus, an event that triggers STING assembly with the downstream enzyme TANK-binding kinase 1 (TBK1). This assembly leads to the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), which in turn induces expression of type-I interferon (IFN) and chemokine genes. STING also mediates inflammatory responses independently of IRF3, but these molecular pathways are largely unexplored. Here, we analyzed the RAW264.7 macrophage secretome to comprehensively identify proinflammatory factors released into the extracellular medium upon STING activation. In total, we identified 1299 proteins in macrophage culture supernatants, of which 23 were significantly increased after STING activation. These proteins included IRF3-dependent cytokines, as well as previously unknown targets of STING, such as the immune semaphorin SEMA4D/CD100, which possesses proinflammatory cytokine-like activities. Unlike for canonical cytokines, the expression of the SEMA4D gene was not up-regulated. Instead, upon STING activation, membrane-bound SEMA4D was cleaved into a soluble form, suggesting the presence of a post-translational shedding machinery. Importantly, the SEMA4D shedding was blocked by TMI-1, an inhibitor of the sheddase ADAM metallopeptidase domain 17 (ADAM17) but not by the TBK1 inhibitor BX795. These results suggest that STING activates ADAM17 and that this activation produces soluble proinflammatory SEMA4D independently of the TBK1/IRF3-mediated transcriptional pathway.
Miyuki Sato, Katsuya Sato, Kotone Tomura, Hidetaka Kosako and Ken Sato : The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans, Nature Cell Biology, Vol.20, No.1, 81-91, 2018.
(要約)
In Caenorhabditis elegans embryos, paternally provided organelles, including mitochondria, are eliminated by a process of selective autophagy called allophagy, the mechanism by which mitochondrial DNA is inherited maternally. However, it remains unclear how paternal organelles are recognized and targeted for autophagy. Here, we identified an autophagy receptor for allophagy, ALLO-1. ALLO-1 is essential for autophagosome formation around paternal organelles and directly binds to the worm LC3 homologue LGG-1 through its LC3-interacting region (LIR) motif. After fertilization, ALLO-1 accumulates on the paternal organelles before autophagosome formation, and this localization depends on the ubiquitin modification of the paternal organelles. We also identified IKKE-1, a worm homologue of the TBK1 and IKKϵ family kinase, as another critical regulator of allophagy. IKKE-1 interacts with ALLO-1, and the IKKE-1-dependent phosphorylation of ALLO-1 is important for paternal organelle clearance. Thus, we propose that ALLO-1 is the allophagy receptor whose function is regulated by IKKE-1-dependent phosphorylation.
Hidetaka Kosako and Kou Motani : Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE, Methods in Molecular Biology, Vol.1487, 137-149, 2017.
Eri Ishikawa, Hidetaka Kosako, Tomoharu Yasuda, Masaki Ohmuraya, Kimi Araki, Tomohiro Kurosaki, Takashi Saito and Sho Yamasaki : Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1., Nature Communications, Vol.7, 12756, 2016.
(要約)
Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4(+)CD8(+) double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1(S557A)) impairs generation of CD4(+) thymocytes. These results suggest that the PKD-SHP-1 axis positively regulates TCR signalling to promote CD4(+) T cell development.
Shiori Akabane, Midori Uno, Naoki Tani, Shunta Shimazaki, Natsumi Ebara, Hiroki Kato, Hidetaka Kosako and Toshihiko Oka : PKA regulates PINK1 stability and Parkin recruitment to damaged mitochondria through phosphorylation of MIC60., Molecular Cell, Vol.62, No.3, 371-384, 2016.
(要約)
A mitochondrial kinase, PTEN-induced putative kinase 1 (PINK1), selectively recruits the ubiquitin ligase Parkin to damaged mitochondria, which modifies mitochondria by polyubiquitination, leading to mitochondrial autophagy. Here, we report that treatment with an adenylate cyclase agonist or expression of protein kinase A (PKA) impairs Parkin recruitment to damaged mitochondria and decreases PINK1 protein levels. We identified a mitochondrial membrane protein, MIC60 (also known as mitofilin), as a PKA substrate. Mutational and mass spectrometric analyses revealed that the Ser528 residue of MIC60 undergoes PKA-dependent phosphorylation. MIC60 transiently interacts with PINK1, and MIC60 downregulation leads to a reduction in PINK1 and mislocalization of Parkin. Phosphorylation-mimic mutants of MIC60 fail to restore the defect in Parkin recruitment in MIC60-knocked down cells, whereas a phosphorylation-deficient MIC60 mutant facilitates the mitochondrial localization of Parkin. Our findings indicate that PKA-mediated phosphorylation of MIC60 negatively regulates mitochondrial clearance that is initiated by PINK1 and Parkin.
Yuki Shindo, Kazunari Iwamoto, Kazunari Mouri, Kayo Hibino, Masaru Tomita, Hidetaka Kosako, Yasushi Sako and Koichi Takahashi : Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling, Nature Communications, Vol.7, 10485, 2016.
(要約)
The phosphorylation cascade in the extracellular signal-regulated kinase (ERK) pathway is a versatile reaction network motif that can potentially act as a switch, oscillator or memory. Nevertheless, there is accumulating evidence that the phosphorylation response is mostly linear to extracellular signals in mammalian cells. Here we find that subsequent nuclear translocation gives rise to a switch-like increase in nuclear ERK concentration in response to signal input. The switch-like response disappears in the presence of ERK inhibitor, suggesting the existence of autoregulatory mechanisms for ERK nuclear translocation involved in conversion from a graded to a switch-like response. In vitro reconstruction of ERK nuclear translocation indicates that ERK-mediated phosphorylation of nucleoporins regulates ERK translocation. A mathematical model and knockdown experiments suggest a contribution of nucleoporins to regulation of the ERK nuclear translocation response. Taken together, this study provides evidence that nuclear translocation with autoregulatory mechanisms acts as a switch in ERK signalling.
Kei Okatsu, Fumika Koyano, Mayumi Kimura, Hidetaka Kosako, Yasushi Saeki, Keiji Tanaka and Noriyuki Matsuda : Phosphorylated ubiquitin chain is the genuine Parkin receptor, The Journal of Cell Biology, Vol.209, No.1, 111-128, 2015.
(要約)
PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson's disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria.
(キーワード)
Amino Acid Sequence / HeLa Cells / Humans / ミトコンドリア (mitochondria) / Molecular Sequence Data / リン酸化 (phosphorylation) / Polyubiquitin / Protein Binding / Protein Kinases / Protein Processing, Post-Translational / Protein Transport / Ubiquitin-Protein Ligases
Fumika Koyano, Kei Okatsu, Hidetaka Kosako, Yasushi Tamura, Etsu Go, Mayumi Kimura, Yoko Kimura, Hikaru Tsuchiya, Hidehito Yoshihara, Takatsugu Hirokawa, Toshiya Endo, Fon A. Edward, Trempe Jean-Francois, Saeki Yasushi, Keiji Tanaka and Noriyuki Matsuda : Ubiquitin is phosphorylated by PINK1 to activate Parkin., Nature, Vol.510, No.7503, 162-166, 2014.
(要約)
PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.
Haruko Tsurumi, Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Kenji Hayashi, Atsuko Matsunaga, Yuko Kajiho, Shoichiro Kanda, Kenichiro Miura, Takashi Sekine, Akira Oka, Kiyonobu Ishizuka, Shigeru Horita, Motoshi Hattori, Seisuke Hattori and Takashi Igarashi : Epithelial protein lost in neoplasm modulates platelet-derived growth factor-mediated adhesion and motility of mesangial cells., Kidney International, Vol.86, No.3, 548-557, 2014.
(要約)
Mesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK-ERK (extracellular signal-regulated kinase) cascade regulated EPLIN-paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor-induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo. These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis.Kidney International advance online publication, 2 April 2014; doi:10.1038/ki.2014.85.
Masahiro Iguchi, Yuki Kujuro, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Norihiro Suzuki, Shinichiro Uchiyama, Keiji Tanaka and Noriyuki Matsuda : Parkin-catalyzed ubiquitin-ester transfer is triggered by PINK1-dependent phosphorylation., The Journal of Biological Chemistry, Vol.288, No.30, 22019-22032, 2013.
(要約)
PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.
Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.
Dysfunction of PINK1, a mitochondrial Ser/Thr kinase, causes familial Parkinson's disease (PD). Recent studies have revealed that PINK1 is rapidly degraded in healthy mitochondria but accumulates on the membrane potential (ΔΨm)-deficient mitochondria, where it recruits another familial PD gene product, Parkin, to ubiquitylate the damaged mitochondria. Despite extensive study, the mechanism underlying the homeostatic control of PINK1 remains unknown. Here we report that PINK1 is autophosphorylated following a decrease in ΔΨm and that most disease-relevant mutations hinder this event. Mass spectrometric and mutational analyses demonstrate that PINK1 autophosphorylation occurs at Ser228 and Ser402, residues that are structurally clustered together. Importantly, Ala mutation of these sites abolishes autophosphorylation of PINK1 and inhibits Parkin recruitment onto depolarized mitochondria, whereas Asp (phosphorylation-mimic) mutation promotes mitochondrial localization of Parkin even though autophosphorylation was still compromised. We propose that autophosphorylation of Ser228 and Ser402 in PINK1 is essential for efficient mitochondrial localization of Parkin.
(キーワード)
Amino Acid Sequence / Animals / HeLa Cells / Humans / Membrane Potentials / Mice / Mitochondria / Molecular Sequence Data / Parkinson Disease / Phosphorylation / Protein Kinases / Protein Transport / Sequence Alignment / Ubiquitin-Protein Ligases
Hidetaka Kosako and Kohji Nagano : Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways, Expert Review of Proteomics, Vol.8, No.1, 81-94, 2011.
(要約)
Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.
(キーワード)
Animals / Electrophoresis, Gel, Two-Dimensional / Evaluation Studies as Topic / Gene Expression Profiling / Humans / Mice / Phosphoproteins / Protein Kinases / Proteomics / Signal Transduction / Tandem Mass Spectrometry
Hidetaka Kosako and Naoko Imamoto : Phosphorylation of nucleoporins: signal transduction-mediated regulation of their interaction with nuclear transport receptors, Nucleus, Vol.1, No.4, 309-313, 2010.
(要約)
The nuclear pore complex (NPC) is composed of ∼30 unique proteins, collectively referred to as nucleoporins or Nups. While metazoan Nups are known to be phosphorylated during mitosis to cause disassembly of the NPC, what is less clear is whether Nups are phosphorylated and regulated by extracellular stimuli in interphase cells. Our multi-step phosphoproteomic approach revealed a number of physiologically relevant extracellular signal-regulated kinase (ERK) targets, including Nups containing FG repeats (FG Nups) that provide binding sites for nuclear transport receptors (NTRs) during the NPC passage. The phosphorylation of FG Nups by ERK does not affect the overall architecture of the NPC but directly inhibits their interactions with NTRs and regulates the permeability barrier properties of the NPC. Such regulation at the levels of transport machinery is expected to have a broad impact on cellular physiology through the spatiotemporal control of signaling events. Until recently, many studies have focused on cellular signaling-mediated phosphorylation of individual cargo proteins, such as transcription factors. An understanding of the effects of signaling pathways on nucleocytoplasmic transport machinery is only beginning to emerge.
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Tohru Tezuka, Takashi Sekine, Takashi Igarashi, Ikuroh Ohsawa, Shigeo Ohta and Seisuke Hattori : Phosphorylation of Nephrin triggers Ca2+ signaling by recruitment and activation of phospholipase C-γ1, The Journal of Biological Chemistry, Vol.284, No.13, 8951-8962, 2009.
(要約)
A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
Seisuke Hattori, Naoyuki Iida and Hidetaka Kosako : Identification of protein kinase substrates by proteomic approaches., Expert Review of Proteomics, Vol.5, No.3, 497-505, 2008.
(要約)
This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.
(キーワード)
Chromatography, Liquid / Electrophoresis, Gel, Two-Dimensional / Phosphorylation / Protein Kinases / Proteomics / Substrate Specificity / Tandem Mass Spectrometry
Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Tohru Tezuka, Takashi Sekine, Takashi Igarashi and Seisuke Hattori : Neph1, a component of the kidney slit diaphragm, is tyrosine phosphorylated by the Src family tyrosine kinase and modulates intracellular signaling by binding to Grb2., The Journal of Biological Chemistry, Vol.283, No.14, 9177-9186, 2008.
(要約)
There are several lines of evidence that the podocyte slit diaphragm (SD), which serves as a structural framework for the filtration barrier in kidney glomerulus, also plays an essential role as a signaling platform. Several SD components including nephrin and TRPC6 are known to be phosphorylated by a Src family tyrosine kinase (SFK), Fyn. Here we have characterized Neph1, another SD component, as a novel substrate of SFK. Fyn interacts with and phosphorylates the cytoplasmic domain of Neph1 in vitro and in intact cells. Peptide mass fingerprinting and site-directed mutagenesis identified several tyrosine phosphorylation sites. In pull-down assays using rat glomerular lysates, Neph1 but not nephrin specifically binds to adaptor protein Grb2 and tyrosine kinase Csk in a phosphorylation-dependent manner. Both tyrosine 637 and 638 of Neph1 are crucial for Neph1-Grb2 binding. Phosphorylation of tyrosine 637 is significantly up-regulated in in vivo models of podocyte injury. Furthermore, Neph1 attenuates ERK activation elicited by Fyn, and this inhibitory effect requires the intact binding motif for the Grb2 SH2 domain. Our results shown here demonstrate that Neph1 is a novel in vivo substrate of SFK and suggest that Neph1 modulates ERK signaling through phosphorylation-dependent interaction with Grb2. Thus, SFK orchestrates a wide spectrum of protein-protein interactions and intracellular signaling networks at SD through tyrosine phosphorylation.
Mei-Ying Han, Hidetaka Kosako, Toshiki Watanabe and Seisuke Hattori : Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN., Molecular and Cellular Biology, Vol.27, No.23, 8190-8204, 2007.
(要約)
Extracellular signal-regulated kinase (ERK) is important for various cellular processes, including cell migration. However, the detailed molecular mechanism by which ERK promotes cell motility remains elusive. Here we characterize epithelial protein lost in neoplasm (EPLIN), an F-actin cross-linking protein, as a novel substrate for ERK. ERK phosphorylates Ser360, Ser602, and Ser692 on EPLIN in vitro and in intact cells. Phosphorylation of the C-terminal region of EPLIN reduces its affinity for actin filaments. EPLIN colocalizes with actin stress fibers in quiescent cells, and stimulation with platelet-derived growth factor (PDGF) induces stress fiber disassembly and relocalization of EPLIN to peripheral and dorsal ruffles, wherein phosphorylation of Ser360 and Ser602 is observed. Phosphorylation of these two residues is also evident during wound healing at the leading edge of migrating cells. Moreover, expression of a non-ERK-phosphorylatable mutant, but not wild-type EPLIN, prevents PDGF-induced stress fiber disassembly and membrane ruffling and also inhibits wound healing and PDGF-induced cell migration. We propose that ERK-mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhanced cell motility.
(キーワード)
Actins / Animals / Cell Movement / Cell Surface Extensions / Cell Survival / Cytoskeletal Proteins / Extracellular Signal-Regulated MAP Kinases / Fibroblasts / HeLa Cells / Humans / Mice / Mitogen-Activated Protein Kinases / NIH 3T3 Cells / Osteoblasts / Phosphoproteins / Phosphorylation / Phosphoserine / Platelet-Derived Growth Factor / Protein Transport / RNA, Small Interfering / Stress Fibers
Michimoto Kobayashi, Takuya Katagiri, Hidetaka Kosako, Naoyuki Iida and Seisuke Hattori : Global analysis of dynamic changes in lipid raft proteins during T-cell activation., Electrophoresis, Vol.28, No.12, 2035-2043, 2007.
(要約)
Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.
Mitsuyo Machida, Hidetaka Kosako, Kyoko Shirakabe, Michimoto Kobayashi, Masato Ushiyama, Junichi Inagawa, Joe Hirano, Tomoyo Nakano, Yasuhiko Bando, Eisuke Nishida and Seisuke Hattori : Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis., The FEBS Journal, Vol.274, No.6, 1576-1587, 2007.
(要約)
Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.
Koji Ueda, Hidetaka Kosako, Yasuhisa Fukui and Seisuke Hattori : Proteomic identification of Bcl2-associated athanogene 2 as a novel MAPK-activated protein kinase 2 substrate., The Journal of Biological Chemistry, Vol.279, No.40, 41815-41821, 2004.
(要約)
The p38 MAPK cascade is activated by various stresses or cytokines. Downstream of p38 MAPKs, there are diversification and extensive branching of signaling pathways. Fluorescent two-dimensional difference gel electrophoresis of phosphoprotein-enriched samples from HeLa cells in which p38 MAPK activity was either suppressed or activated enabled us to detect approximately 90 candidate spots for factors involved in p38-dependent pathways. Among these candidates, here we identified four proteins including Bcl-2-associated athanogene 2 (BAG2) by peptide mass fingerprintings. BAG family proteins are highly conserved throughout eukaryotes and regulate Hsc/Hsp70-mediated molecular chaperone activities and apoptosis. The results of two-dimensional immunoblots suggested that the phosphorylation of BAG2 was specifically controlled in a p38 MAPK-dependent manner. Furthermore, BAG2 was directly phosphorylated at serine 20 in vitro by MAPK-activated protein kinase 2 (MAPKAP kinase 2), which is known as a primary substrate of p38 MAPK and mediates several p38 MAPK-dependent processes. We confirmed that MAPKAP kinase 2 is also required for phosphorylation of BAG2 in vivo. Thus, p38 MAPK-MAPKAP kinase 2-BAG2 phosphorylation cascade may be a novel signaling pathway for response to extracellular stresses.
(キーワード)
Electrophoresis, Gel, Two-Dimensional / HSP70 Heat-Shock Proteins / HeLa Cells / Humans / Intracellular Signaling Peptides and Proteins / MAP Kinase Signaling System / Mitogen-Activated Protein Kinases / Molecular Chaperones / Phosphoproteins / Phosphorylation / Protein-Serine-Threonine Kinases / Proteomics / Serine / p38 Mitogen-Activated Protein Kinases
Shin-ichi Yamamoto, Motoshi Nagao, Michiya Sugimori, Hidetaka Kosako, Hirofumi Nakatomi, Naoya Yamamoto, Hirohide Takebayashi, Yo-ichi Nabeshima, Toshio Kitamura, Gerry Weinmaster, Kozo Nakamura and Masato Nakafuku : Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord., The Journal of Neuroscience, Vol.21, No.24, 9814-9823, 2001.
(要約)
Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.
Rumiko Mizuguchi, Michiya Sugimori, Hirohide Takebayashi, Hidetaka Kosako, Motoshi Nagao, Shosei Yoshida, Yo-ichi Nabeshima, Kenji Shimamura and Masato Nakafuku : Combinatorial roles of Olig2 and Neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons., Neuron, Vol.31, No.5, 757-771, 2001.
(要約)
Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.
Hidetaka Kosako, Toshimichi Yoshida, Fumio Matsumura, Toshimasa Ishizaki, Shuh Narumiya and Masaki Inagaki : Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow., Oncogene, Vol.19, No.52, 6059-6064, 2000.
(要約)
The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.
Hirohide Takebayashi, Shosei Yoshida, Michiya Sugimori, Hidetaka Kosako, Ryo Kominami, Masato Nakafuku and Yo-ichi Nabeshima : Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3., Mechanisms of Development, Vol.99, No.1-2, 143-148, 2000.
(要約)
Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.
Hidemasa Goto, Hidetaka Kosako and Masaki Inagaki : Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase., Microscopy Research and Technique, Vol.49, No.2, 173-182, 2000.
(要約)
Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis.
Hidemasa Goto, Yasuko Tomono, Kozo Ajiro, Hidetaka Kosako, Masatosi Fujita, Minoru Sakurai, Katsuya Okawa, Akihiro Iwamatsu, Tohru Okigaki, Toshitada Takahashi and Masaki Inagaki : Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation., The Journal of Biological Chemistry, Vol.274, No.36, 25543-25549, 1999.
(要約)
Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.
Ryota Hashimoto, Yu Nakamura, Hidetaka Kosako, Mutsuki Amano, Kozo Kaibuchi, Masaki Inagaki and Masatoshi Takeda : Distribution of Rho-kinase in the bovine brain., Biochemical and Biophysical Research Communications, Vol.263, No.2, 575-579, 1999.
73.
Hidetaka Kosako, Hidemasa Goto, Maki Yanagida, Kaori Matsuzawa, Masatoshi Fujita, Yasuko Tomono, Tohru Okigaki, Hideharu Odai, Kozo Kaibuchi and Masaki Inagaki : Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments., Oncogene, Vol.18, No.17, 2783-2788, 1999.
74.
Hidemasa Goto, Hidetaka Kosako, Kazushi Tanabe, Maki Yanagida, Minoru Sakurai, Mutsuki Amano, Kozo Kaibuchi and Masaki Inagaki : Phosphorylation of vimentin by Rho-associated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis., The Journal of Biological Chemistry, Vol.273, No.19, 11728-11736, 1998.
75.
Hidetaka Kosako, Mutsuki Amano, Maki Yanagida, Kazushi Tanabe, Yoshimi Nishi, Kozo Kaibuchi and Masaki Inagaki : Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase., The Journal of Biological Chemistry, Vol.272, No.16, 10333-10336, 1997.
76.
Kaori Matsuzawa, Hidetaka Kosako, Naoyuki Inagaki, Hideki Shibata, Hideyuki Mukai, Yoshitaka Ono, Mutsuki Amano, Kozo Kaibuchi, Yoshiharu Matsuura, Ichiro Azuma and Masaki Inagaki : Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN., Biochemical and Biophysical Research Communications, Vol.234, No.3, 621-625, 1997.
77.
Hidetaka Kosako, Yukiko Gotoh and Eisuke Nishida : Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis., Development Growth & Differentiation, Vol.38, 577-582, 1996.
78.
Hidetaka Kosako, Yoshiko Akamatsu, Naoya Tsurushita, Lee Kyung-Kwon, Yukiko Gotoh and Eisuke Nishida : Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries., Biochemistry, Vol.35, No.40, 13212-13221, 1996.
79.
Makoto Fukuda, Yukiko Gotoh, Hidetaka Kosako, Seisuke Hattori and Eisuke Nishida : Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes., The Journal of Biological Chemistry, Vol.269, No.52, 33097-33101, 1994.
80.
Hidetaka Kosako, Yukiko Gotoh and Eisuke Nishida : Mitogen-activated protein kinase kinase is required for the Mos-induced metaphase arrest., The Journal of Biological Chemistry, Vol.269, No.45, 28354-28358, 1994.
Hidetaka Kosako, Yukiko Gotoh and Eisuke Nishida : Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation., The EMBO Journal, Vol.13, No.9, 2131-2138, 1994.
83.
Hidetaka Kosako, Yukiko Gotoh and Eisuke Nishida : Regulation and function of the MAP kinase cascade in Xenopus oocytes., Journal of Cell Science, Vol.18, 115-119, 1994.
84.
Hidetaka Kosako, Eisuke Nishida and Yukiko Gotoh : cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates., The EMBO Journal, Vol.12, No.2, 787-794, 1993.
85.
Kazuyuki Tobe, Takashi Kadowaki, Kenta Hara, Yukiko Gotoh, Hidetaka Kosako, Satoshi Matsuda, Hiroyuki Tamemoto, Kohjiro Ueki, Yasuo Akanuma, Eisuke Nishida and Yoshio Yazaki : Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection., The Journal of Biological Chemistry, Vol.267, No.29, 21089-21097, 1992.
86.
Hidetaka Kosako, Yukiko Gotoh, Satoshi Matsuda, Satoshi Matsuda and Eisuke Nishida : Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation., The EMBO Journal, Vol.11, No.8, 2903-2908, 1992.
87.
Satoshi Matsuda, Hidetaka Kosako, Katsuya Takenaka, Kenji Moriyama, Hikoichi Sakai, Tetsu Akiyama, Yukiko Gotoh and Eisuke Nishida : Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade., The EMBO Journal, Vol.11, No.3, 973-982, 1992.
88.
Yukiko Gotoh, Eisuke Nishida, Satoshi Matsuda, Nobuyuki Shiina, Hidetaka Kosako, Koichiro Shiokawa, Tetsu Akiyama, Kunihiro Ohta and Hikoichi Sakai : In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase., Nature, Vol.349, No.6306, 251-254, 1991.
(要約)
The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.
Shunya Yamauchi, Noriko Saito-Tarashima, Kou Motani, Hidetaka Kosako and Noriaki Minakawa : Synthesis of cyclic dinucleotide analog enhanced membrane permeability, 15h International Symposium on Nanomedicine (ISNM2022), Dec. 2022.
2.
Hidetaka Kosako : Advanced proteomic approaches to elucidate disease-related signaling mechanisms., The 5th Symposium of the Inter-University Research Network for Trans-Omics Medicine, Online,, Jan. 2021.
3.
Kou Motani and Hidetaka Kosako : BioID-based Screening Of Biotinylation Sites Using Tamavidin 2-REV Globally Identifies Interactors Of Stimulator Of Interferon Genes (STING), ASCB | EMBO 2019 Meeting, Washington DC, USA,, Dec. 2019.
4.
Izumi Ohigashi, Yu Tanaka, Kenta Kondou, Sayumi Fujimori, Amy C. Palin, Hiroyuki Kondo, Hidetaka Kosako and Yousuke Takahama : Trans-omics impact of thymoproteasome in cortical thymic epithelial cells, ThymE: T cell and thymus biology, May 2019.
5.
Kou Motani and Hidetaka Kosako : BioID-Based Screening of Biotinylation Sites Globally Identifies STING Interactors, Keystone Symposia "Proteomics and its Application to Translational and Precision Medicine, Stockholm, Sweden,, Apr. 2019.
6.
Yuichi Takashi, Yuka Kinoshita, Nobuaki Ito, Shun Sawatsubashi, Hidetaka Kosako, Masahiro Abe, Munehide Matsuhisa, Toshio Matsumoto and Seiji Fukumoto : FGF receptor 1c works as a phosphate-sensor to regulate FGF23 production, ASBMR 2018 Annual Meeting Registration Confirmation, Sep. 2018.
7.
Kou Motani, Mayumi Kajimoto and Hidetaka Kosako : Identification of the ATG8 family member GABARAPL2 as a novel TBK1 substrate, 12th International Symposium of the Institute Network, Nov. 2017.
8.
Hidetaka Kosako, Megumi Kawano, Keiji Tanaka and Noriyuki Matsuda : Quantitative interactome and phosphoproteome analysis identifies novel signaling components of Parkinson's disease-associated kinase PINK1., HUPO2017, Dublin, Ireland,, Sep. 2017.
9.
Hidetaka Kosako, Eri Ishikawa and Sho Yamasaki : Dissection of protein kinase D signaling during thymocyte development using various phosphoproteomic strategies., HUPO 2016, Taipei, Taiwan,, Sep. 2016.
10.
Hidetaka Kosako : Identification and Functional Analysis of Protein Kinase Substrates using Various Proteomic Technologies, Keystone Symposia "The Biological Code of Cell Signaling: A Tribute to Tony Pawson", Colorado, USA, Jan. 2015.
11.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Hisaaki Taniguchi and Naoko Imamoto : Multisite phosphorylation of FG nucleoporins by MAP kinases is involved in the regulation of nucleocytoplasmic transport, Cold Spring Harbor Laboratory Meeting "Dynamic Organization of Nuclear Function", Cold Spring Harbor, USA, Sep. 2012.
12.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Multisite phosphorylation of nucleoporins by ERK and p38 MAP kinases is implicated in the regulation of nuclear transport, Gordon Research Conference "Phosphorylation & G-Protein Mediated Signaling Networks", Biddeford, USA, Jun. 2012.
13.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Phosphorylation of FG-repeat nucleoporins by MAP kinases is implicated in the control of nuclear transport, EMBO Conference "Cellular Signaling & Molecular Medicine", Dubrovnik, Croatia, May 2012.
14.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Phosphorylation of FG nucleoporins by ERK and p38 MAP kinases is involved in the regulation of nucleocytoplasmic transport, 2011 American Society for Cell Biology Annual Meeting, Denver, USA, Dec. 2011.
15.
Hidetaka Kosako, Shigehiro Yoshimura, Megumi Kawano, Shingo Kose and Naoko Imamoto : Phosphorylation of multiple FG nucleoporins by MAP kinases is involved in the regulation of nucleocytoplasmic transport, Keystone Symposia "Omics Meets Cell Biology", Alpbach, Austria, May 2011.
16.
Hidetaka Kosako : Phosphoproteomics reveals ERK MAP kinase-mediated regulation of nuclear pore complex proteins, BIT's 3rd World Cancer Congress, Singapore, Jun. 2010.
17.
Hidetaka Kosako, Chizuru Aranami, Nozomi Yamaguchi, Hitomi Suzuki, Shingo Kose, Naoko Imamoto, Hisaaki Taniguchi, Eisuke Nishida and Seisuke Hattori : Phosphorylation of nuclear pore complex proteins by ERK MAP kinase regulates interaction with transport receptors, 49th Annual Meeting of American Society for Cell Biology, San Diego, USA, Dec. 2009.
Kou Motani, Noriko Saito-Tarashima, K Nishino, Shunya Yamauchi, Noriaki Minakawa and Hidetaka Kosako : ACBD3 forms specialized ER-Golgi contact sites to drive the ER exit of STING., The 17th International Symposium of the Institute Network, Kanazawa, Oct. 2022.
6.
Tetsuro Yoshimaru, Yosuke Matsushita, Hidetaka Kosako, Sasa Mitsunorii, Miyoshi Yasuo and Toyomasa Katagiri : The plasma membrane BIG3-PHB2 complex contributes to the acquisition of trastuzumab-resistance in HER2-positive breast cancer, The 17th International Symposium of the Institute Network for Biomedical Sciences International Symposium on Tumor Biology in Kanazawa 2022, Oct. 2022.
7.
𠮷川 治孝, 小迫 英尊 : Protein Correlation Profilingによる細胞内巨大タンパク質複合体の解析, 日本プロテオーム学会2022年大会, 2022年8月.
髙士 祐一, 小迫 英尊, 沢津橋 俊, Y Kinoshita, N Ito, 松久 宗英, M Matsumoto, 福本 誠二 : FGF recepotor I works as a phosphate-sensor to regulate FGF23 production, 15th Bone Biology Forum, 2019年8月.