Poster

Poster 9 Cell Signaling 2 ポスター 9 細胞内外のシグナル2

P9-1 MARK4 is activated via its phosphorylation by Cdk5 タウキナーゼMARK4はCdk5によるリン酸化で活性化する Saito Taro（斎藤太郎），大場俊弥，安藤香奈絵 Dept Biol. Sci, Tokyo Met Univ Microtubule-associated protein tau is phosphorylated by multiple protein kinases. However, their relationships are not well known yet. Microtubule affinity-regulating kinase (MARK) and cyclin-dependent kinase 5 (Cdk5) are well known as tau kinases in not only physiological but also pathological conditions. MARK is activated by its phosphorylation in the activation loop by LKB1. Here, we report that MARK4, a member of MARK family (MARK1-4), was activated by Cdk5 via its direct phosphorylation. When phosphorylation of tau by MARK4 was examined in transfected HEK293 cells, co-expression of Cdk5-p35 dramatically enhanced the phosphorylation of tau by MARK4 in MARK sites (Ser262 and Ser356), whereas MARK2, another member of MARK family, was not. The activation of MARK4 by Cdk5 was also examined by in vitro kinase assay. Cdk5 did not affect the phosphorylation of MARK4 by LKB1. Since there is a putative phosphorylation site for Cdk5 in the activation loop of MARK4 (Ser218) nearby the LKB1 site (Thr214), the kinase activity of a mutant MARK4, whose Ser218 is substituted by Ala, was investigated. The mutant MARK4 lost its kinase activity with or without Cdk5, suggesting that the phosphorylation of Ser218 might be essential for the activation of MARK4. These results indicate that MARK4 has unique activation mechanisms in MARK family.		P9-2 Tyrosine phosphorylation of SIRPα as a cellular stress response correlated with intracellular Cl^- dynamics 細胞内クロライドと関連するストレス応答反応としてのSIRPαチロシンリン酸化 Jingu Daiki（神宮大輝）¹，飯野美香¹，橋本美穂¹，草苅伸也¹，今井香織¹，的崎尚²，大西浩史¹ ¹Dept. Lab. Sci., Gunma Univ. Grad. Sch. Health Sci., Gunma, Japan ²Dept. Biochem. Mol. Biol., Kobe Univ. Grad. Sch. Med., Kobe, Japan SIRPα (Signal regulatory protein α) is a member of immunoglobulin superfamily membrane proteins that is tyrosine phosphorylated in its cytoplasmic region, and thereby binds and activates a cytoplasmic protein tyrosine phosphatase, Shp2. In the central nervous system, SIRPα is expressed in matured neurons, as well as in microglia. Although physiological function of SIRPα has not been fully understood in the central nervous system, SIRPα undergoes tyrosine phosphorylation in the brain of mice in response to forced swim stress in cold water, and this signaling mechanism is implicated in regulation of the behavior (immobility) of mice during the forced swim. Lowered body temperature in the cold water is the primary cause of the induction of tyrosine phosphorylation of SIRPα in the brain, because low temperature directly induces tyrosine phosphorylation of SIRPα in cultured cells. We found that Cl^- depletion is another cellular stress that induced tyrosine phosphorylation of SIRPα in microglia model cell line (MG6) and primary cultured neurons. Cl^- channel blockers suppressed the Cl^- depletion-induced tyrosine phosphorylation SIRPα. Furthermore, low temperature-induced phosphorylation of SIRPα was also suppressed by the treatment. Intracellular Cl^- concentration was inversely correlated to the phosphorylation of SIRPα. Cl^- dynamics is thus an important factor for tyrosine phosphorylation of SIRPα.

P9-3 PRMT1 regulates COPI vesicle transport via methylation of Scyl1 at cis-Golgi シスゴルジにおいてPRMT1はScyl1のメチル化を介してCOPI小胞輸送を制御する Amano Genki（天野元揮）¹，松崎伸介^1,2，森泰丈³，高村明孝¹，韓薩日娜¹，鹿田星¹，三好耕¹，吉村武¹，片山泰一¹ ¹Molecular Brain Science, United Graduate School of Child Development, Osaka University ²Department of Pharmacology, Wakayama Medical University ³Department of Anatomy and Neuroscience, Graduate School of Medicine, International University of Health and Welfare Cumulative of reports indicate that the cellular events in response to endoplasmic reticulum(ER) stress should relate to the pathology of neurodegenerative diseases, such as Alzheimer’s disease. On the other hands, previous studies have reported that Protein arginine N-methyltransferase 1 (PRMT1) promotes expression of molecular chaperones under ER stress. PRMT1 accounts for about 85% of arginine methylation in cells and is known to be involved in the regulation of transcription factors and cell proliferation. However, the functions of PRMT1 in cytoplasm are still unknown. We examined the importance of PRMT1 in organelles homeostasis in cytoplasm. We verified the effect of PRMT1 knocked down on organelles. The PRMT1 knock down cells showed abnormal cis-Golgi, but not any remarkable changes of other. Furthermore, there was no change in ER stress level in PRMT1 transient knock down cells, but PRMT1 stable knock down cells were significantly increased ER stress levels. We hypothesized that PRMT1 modified some target proteins at cis-Golgi to regulate ER-Golgi balance. To identify the candidate factors, we analyzed those with methylation level change in microsomal fraction under ER stress condition. As a result, we identified Scyl1. Recently Scyl1 has been reported to function in retrograde COPI-mediated intracellular transport. We showed the effect on the COPI vesicle trafficking by methylation of Scyl1 via PRMT1.		P9-4 Effects of serotonin on the fetal movement-like activity in the spinal cord セロトニンの脊髄における胎動性活動に対する効果 Arata Akiko（荒田晶子）¹，内田千晶¹，外村宗達^1,2，大岡裕隆¹，大村吉幸³ ¹Dept.of Physiology, Hyogo College of Medicine ²Dept. of Anatomy, Hyogo College of Med., Nishinomiya, Hyogo Japan ³Dept. of Mechano-Informatics, School of Information Science and Technology, Univ. of Tokyo Fetal movement-like activity (FMA) is suppressed by strychnine-sensitive glycine receptor after birth (Robinson SR. 2000; Shimomura et al., 2015). Both of respiratory and the FMA recorded from spinal nerve roots are observed not only cervical level but also whole spinal level. The respiratory rhythm generator is located in the medulla and the FMA generator is in the spinal cord; however, the FMA generator is not fully understood. In this study, we examined the location and the properties of the FMA generator. At the First, we compared the responses of isolated whole spinal cord preparation to that of regional spinal cord (cervical: C or thoracic: Th or lumbar: L or sacra: S) preparation under strychnine application. The FMA in whole spinal cord showed more continuously and more frequently than the regional spinal cord. In whole spinal cord, the timing of the FMA in each segment showed synchronously, however, interestingly, the FMA in each C-S regional spinal cord showed independently and produced its own rhythm. It is well known that 5-HT has strongly affected to the spinal activity such as locomotion. We examined effect of 5-Ht on the spinal activity. When we added 5-HT and strychnine applications to these preparations, the FMA in each regional spinal cord showed some regularity. These results suggested that the FMA generator existed in the whole spinal cord, and each spinal region produced their own rhythm even if the spinal cord was cut; The FMA generator can produce own rhythm in segmental preparation and can be accelerated by 5-HT. We demonstrated their origin of FMA and examined how their activity spread by the cycle-triggered-optical imaging method which identified only FMA related activities.

P9-5 Expression of pro-inflammatory cytokines and their receptors in GT1-7 cells GT1-7細胞における炎症性サイトカインとその受容体の発現 Kuwahara-Otani Sachi（大谷佐知），大穂雄太，湊雄介，前田誠司，八木秀司 Dept. of Anat. & Cell Biol., Hyogo College of Med. Gonadotropin-releasing hormone (GnRH) neurons have a central role in the regulation of reproduction. Inflammation and stress can modulate reproduction through the alteration of the hypothalamus function. Several reports indicated that several inflammatory cytokines modulate the hypothalamus function. However, there is limited information on the interaction between cytokines and GnRH neurons. Using immortalized GnRH neurons (GT1-7 cells), we examined the expression of the mRNAs for some inflammatory cytokines, TNF-α, IL-1β, IL-18 and IL-6, and their receptors. These cytokines and their receptors were expressed in GT1-7 cells, suggesting a direct effect of cytokines in the control of the synthesis and/or release of the hormone. To investigate whether TNF-α activates NF-κB in GT1-7 cells, we applied recombinant TNF-α and studied intracellular localization of NF-κB p65 using immunocytochemistry. In untreated cells NF-κB p65 was totally inactive and retained in the cytoplasm. Administration of TNF-α induced a nuclear NF-κB p65 translocation. These results suggest that TNF-α directly effects on GT1-7 cells via activation of the NF-κB pathway and influence neuroendocrine regulation of GnRH neurons.