Faculty Profiles - HISAMOTO, Naoki

Papers - HISAMOTO, Naoki

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A cytidine deaminase regulates axon regeneration by modulating the functions of the Caenorhabditis elegans HGF/plasminogen family protein SVH-1

Shimizu, T; Nomachi, T; Matsumoto, K; Hisamoto, N

PLOS GENETICS Vol. 20 ( 7 ) page： e1011367 2024.7

Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans

Sakai, Y; Shimizu, T; Tsunekawa, M; Hisamoto, N; Matsumoto, K

PLOS GENETICS Vol. 19 ( 12 ) page： e1011089 2023.12

LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site Reviewed

Hanafusa, H; Fujita, K; Kamio, M; Iida, S; Tamura, Y; Hisamoto, N; Matsumoto, K

JOURNAL OF CELL SCIENCE Vol. 136 ( 6 ) 2023.3

Histidine dephosphorylation of the Gβ protein GPB-1 promotes axon regeneration in C. elegans Reviewed

Yoshiki Sakai, Hiroshi Hanafusa, Naoki Hisamoto, Kunihiro Matsumoto

EMBO Reports Vol. 23 ( 12 ) page： e55076 2022.12

The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation Reviewed

Fujita, K; Kedashiro, S; Yagi, T; Hisamoto, N; Matsumoto, K; Hanafusa, H

JOURNAL OF CELL SCIENCE Vol. 135 ( 23 ) 2022.12

LRRK1-mediated NDEL1 phosphorylation promotes cilia disassembly via dynein-2-driven retrograde intraflagellar transport Reviewed

Hanafusa, H; Kedashiro, S; Gotoh, M; Saitoh, KH; Inaba, H; Nishioka, T; Kaibuchi, K; Inagaki, M; Hisamoto, N; Matsumoto, K

JOURNAL OF CELL SCIENCE Vol. 135 ( 21 ) 2022.11

Chemical Signaling Regulates Axon Regeneration via the GPCR-Gqa Pathway in Caenorhabditis elegans Reviewed International coauthorship

Shimizu Tatsuhiro, Sugiura Kayoko, Sakai Yoshiki, Dar Abdul R., Butcher Rebecca A., Matsumoto Kunihiro, Hisamoto Naoki

JOURNAL OF NEUROSCIENCE Vol. 42 ( 5 ) page： 720 - 730 2022.2

A Microfluidic Device with Check Valves to Detect Cancer using Caenorhabditis elegans

Shiga, H; Takeuchi, M; Kim, E; Hisamoto, N; Ishikawa, T; Fukuda, T

2022 IEEE/SICE INTERNATIONAL SYMPOSIUM ON SYSTEM INTEGRATION (SII 2022) page： 969 - 970 2022

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons Reviewed International coauthorship

Kulkarni, SS; Sabharwal, V; Sheoran, S; Basu, A; Matsumoto, K; Hisamoto, N; Ghosh-Roy, A; Koushika, SP

GENETICS Vol. 219 ( 3 ) 2021.11

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation Reviewed

Okumura, F; Oki, N; Fujiki, Y; Ikuta, R; Osaki, K; Hamada, S; Nakatsukasa, K; Hisamoto, N; Hara, T; Kamura, T

SCIENTIFIC REPORTS Vol. 11 ( 1 ) page： 20880 2021.10

CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner Reviewed

Hisamoto, N; Sakai, Y; Ohta, K; Shimizu, T; Li, C; Hanafusa, H; Matsumoto, K

JOURNAL OF NEUROSCIENCE Vol. 41 ( 40 ) page： 8309 - 8320 2021.10

The Integrin Signaling Network Promotes Axon Regeneration via the Src-Ephexin-RhoA GTPase Signaling Axis Reviewed

Sakai, Y; Tsunekawa, M; Ohta, K; Shimizu, T; Pastuhov, SI; Hanafusa, H; Hisamoto, N; Matsumoto, K

JOURNAL OF NEUROSCIENCE Vol. 41 ( 22 ) page： 4754 - 4767 2021.6

BRCA1-BARD1 Regulates Axon Regeneration in Concert with the Gqα-DAG Signaling Network Reviewed

Sakai Y, Hanafusa H, Shimizu T, Pastuhov SI, Hisamoto N, Matsumoto K.

Journal of Neuroscience Vol. 41 ( 13 ) page： 2842 - 2853 2021.3

Caenorhabditis elegans F-Box Protein Promotes Axon Regeneration by Inducing Degradation of the Mad Transcription Factor Reviewed

Shimizu T, Pastuhov SI, Hanafusa H, Sakai Y, Todoroki Y, Hisamoto N, Matsumoto K.

Journal of Neuroscience Vol. 41 ( 11 ) page： 2373 - 2381 2021.3

Engulfment Genes Promote Neuronal Regeneration inCaenorhabditis Elegans: Two Divergent But Complementary Views Invited Reviewed International coauthorship

Chang Chieh, Hisamoto Naoki

BIOESSAYS Vol. 42 ( 8 ) page： e1900185 2020.8

Factors regulating axon regeneration via JNK MAP kinase in Caenorhabditis elegans Invited Reviewed

JOURNAL OF BIOCHEMISTRY Vol. 167 ( 5 ) page： 433 - 439 2020.5

TDP2 negatively regulates axon regeneration by inducing SUMOylation of an Ets transcription factor

Sakai Yoshiki, Hanafusa Hiroshi, Pastuhov Strahil Iv, Shimizu Tatsuhiro, Li Chun, Hisamoto Naoki, Matsumoto Kunihiro

EMBO REPORTS Vol. 20 ( 10 ) page： e47517 2019.10

N-Glycosylation of the Discoidin Domain Receptor Is Required for Axon Regeneration in Caenorhabditis elegans

Shimizu Tatsuhiro, Kato Yuka, Sakai Yoshiki, Hisamoto Naoki, Matsumoto Kunihiro

GENETICS Vol. 213 ( 2 ) page： 491 - 500 2019.10

C. elegans Tensin Promotes Axon Regeneration by Linking the Met-like SVH-2 and Integrin Signaling Pathways Reviewed

Hisamoto Naoki, Shimizu Tatsuhiro, Asai Kazuma, Sakai Yoshiki, Pastuhov Strahil I, Hanafusa Hiroshi, Matsumoto Kunihiro

JOURNAL OF NEUROSCIENCE Vol. 39 ( 29 ) page： 5662 - 5672 2019.7

LRRK1 phosphorylation of Rab7 at S72 links trafficking of EGFR-containing endosomes to its effector RILP Reviewed

Hanafusa Hiroshi, Yagi Takuya, Ikeda Haruka, Hisamoto Naoki, Nishioka Tomoki, Kaibuchi Kozo, Shirakabe Kyoko, Matsumoto Kunihiro

JOURNAL OF CELL SCIENCE Vol. 132 ( 11 ) 2019.6

The C. elegans BRCA2-ALP/Enigma Complex Regulates Axon Regeneration via a Rho GTPase-ROCK-MLC Phosphorylation Pathway Reviewed

Shimizu Tatsuhiro, Pastuhov Strahil Iv, Hanafusa Hiroshi, Matsumoto Kunihiro, Hisamoto Naoki

Cell reports Vol. 24 ( 7 ) page： 1880 - 1889 2018.8

Phosphatidylserine exposure mediated by ABC transporter activates the integrin signaling pathway promoting axon regeneration Reviewed

Hisamoto Naoki, Tsuge Anna, Pastuhov Strahil Iv, Shimizu Tatsuhiro, Hanafusa Hiroshi, Matsumoto Kunihiro

NATURE COMMUNICATIONS Vol. 9 ( 1 ) page： 3099 2018.8

UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes

Choudhary Bikash, Kamak Madhushree, Ratnakaran Neena, Kumar Jitendra, Awasthi Anjali, Li Chun, Nguyen Ken, Matsumoto Kunihiro, Hisamoto Naoki, Koushika Sandhya P.

PLOS GENETICS Vol. 13 ( 11 ) page： e1007100 2017.11

Signal transduction cascades in axon regeneration: insights from C. elegans

Hisamoto Naoki, Matsumoto Kunihiro

CURRENT OPINION IN GENETICS & DEVELOPMENT Vol. 44 page： 54 - 60 2017.6

Higher Precision Rotational Manipulation of C. elegans by Microchannel

Nakajima Masahiro, Igarashi Yu, Takeuchi Masaru, Hisamoto Nagoki, Hasegawa Yasuhisa, Fukuda Toshio

2017 IEEE 17TH INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (IEEE-NANO) page： 297 - 298 2017

The C. elegans Discoidin Domain Receptor DDR-2 Modulates the Met-like RTK-JNK Signaling Pathway in Axon Regeneration. Reviewed

Hisamoto N, Nagamori Y, Shimizu T, Pastuhov SI, Matsumoto K.

PLoS Genetics Vol. 12 ( 12 ) page： e1006475 2016.12

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

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2-JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.

DOI： 10.1371/journal.pgen.1006475

The core molecular machinery used for engulfment of apoptotic cells regulates the JNK pathway mediating axon regeneration in Caenorhabditis elegans. Reviewed

Pastuhov, S.I., Fujiki, K., Tsuge, A., Asai, K., Ishikawa, S., Hirose, K., Matsumoto, K., Hisamoto, N.

Journal of Neuroscience Vol. 36 ( 37 ) page： 9710-9721 2016.9

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

The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway.

DOI： 10.1523/JNEUROSCI.0453-16.2016

Axotomy-induced HIF/serotonin signaling axis promotes axon regeneration in C. elegans. Reviewed

Alam T, Maruyama H, Li C, Pastuhov SI, Nix P, Bastiani M, Hisamoto N, Matsumoto K.

NATURE COMMUNICATIONS Vol. 7 page： 10388 2016.1

Axon regeneration is regulated by Ets-C/EBP transcription complexes generated by activation of the cAMP/Ca²⁺-p38 MAPK signaling pathways. Reviewed

Chun Li, Naoki Hisamoto, Kunihiro Matsumoto

PLoS Genetics Vol. 11 page： e1005603 2015.10

The C. elegans HGF/plasminogen-like protein SVH-1 has protease-dependent and -independent functions Reviewed

Naoki Hisamoto, Chun Li, Motoki Yoshida, Kunihiro Matsumoto

Cell reports 2014

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

脊椎動物の肝細胞増殖因子(HGF)とプラスミノーゲンは、それぞれ増殖因子と細胞外マトリクス(ECM)を切断するプロテアーゼという全く異なる機能を持つが、構造およびアミノ酸配列の類似性から、共通の祖先遺伝子から分岐したと考えられている。しかし、これらの因子が２つの機能を同時に持つ祖先遺伝子からそれぞれ機能分化したのか、それとも脊椎動物で増殖因子の機能が後発的に獲得されたのか、その進化的経緯は不明であった。我々は以前、線虫C. エレガンスにおいてHGF/プラスミノーゲン様因子SVH-1がHGF受容体ホモログであるSVH-2を介して神経軸索再生を制御することを報告した。今回、我々はSVH-1およびSVH-2遺伝子が他の無脊椎動物でも広く保存されていること、さらにSVH-1がSVH-2非依存的に幼虫の生育を制御することを見出した。SVH-1は機能的なプロテアーゼドメインを持ち、その活性は生育に必須であるが軸索再生には必須ではない。svh-1遺伝子の欠損はECMタンパク質FBL-1の咽頭筋への異常な蓄積を引き起こす。またsvh-1変異体の幼虫期での生育停止の表現型は、fbl-1遺伝子の欠損により部分的に抑圧された。以上の結果から、SVH-1は増殖因子かつプロテアーゼという２つの異なる機能を持った因子であること、またHGFとプラスミノーゲンは両方の機能を持つ祖先型から分岐した可能性が示唆された。

DOI： http://dx.doi.org/10.1016/j.celrep.2014.10.056

Endocannabinoid-Goα signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signalling Reviewed

Strahil Iv. Pastuhov, Kota Fujiki, Paola Nix, Shuka Kanao, Michael Bastiani, Kunihiro Matsumoto and Naoki Hisamoto

Nature Communications 2012.10

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

The ability of neurons to regenerate their axons after injury is determined by a balance between cellular pathways that promote and those that inhibit regeneration. In Caenorhabditis elegans, axon regeneration is positively regulated by the c-Jun N-terminal kinase mitogen activated protein kinase pathway, which is activated by growth factor-receptor tyrosine kinase signalling. Here we show that fatty acid amide hydrolase-1, an enzyme involved in the degradation of the endocannabinoid anandamide (arachidonoyl ethanolamide), regulates the axon regeneration response of γ-aminobutyric acid neurons after laser axotomy. Exogenous arachidonoyl ethanolamide inhibits axon regeneration via the Goα subunit GOA-1, which antagonizes the Gqα subunit EGL-30. We further demonstrate that protein kinase C functions downstream of Gqα and activates the MLK-1-MEK-1-KGB-1 c-Jun N-terminal kinase pathway by phosphorylating MLK-1. Our results show that arachidonoyl ethanolamide induction of a G protein signal transduction pathway has a role in the inhibition of post-development axon regeneration.

DOI： 10.1038/ncomms2136

C. elegans growth factor-receptor tyrosine kinase signaling regulates axon regeneration. Reviewed

Li C, Hisamoto N, Nix P, Kanao S, Mizuno T, Bastiani M and Matsumoto K.

Nature Neuroscience Vol. 15 ( 4 ) page： 551-557 2012.4

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

The ability of neurons to undergo regenerative growth after injury is governed by cell-intrinsic and cell-extrinsic regeneration pathways. These pathways represent potential targets for therapies to enhance regeneration. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, the Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways are important for axon regeneration. We found that the C. elegans SVH-1 growth factor and its receptor, SVH-2 tyrosine kinase, regulate axon regeneration. Loss of SVH-1-SVH-2 signaling resulted in a substantial defect in the ability of neurons to regenerate, whereas its activation improved regeneration. Furthermore, SVH-1-SVH-2 signaling was initiated extrinsically by a pair of sensory neurons and functioned upstream of the JNK-MAPK pathway. Thus, SVH-1-SVH-2 signaling via activation of the MAPK pathway acts to coordinate neuron regeneration response after axon injury.

DOI： 10.1038/nn.3052

Heavy Metal Stress Assay of Caenorhabditis elegans

Pastuhov Strahil Iv, Shimizu Tatsuhiro, Hisamoto Naoki

BIO-PROTOCOL Vol. 7 ( 11 ) page： e2312 2017.6

Endocannabinoid signaling regulates regenerative axon navigation in Caenorhabditis elegans via the GPCRs NPR-19 and NPR-32. Reviewed

Pastuhov SI, Matsumoto K, Hisamoto N.

Genes to Cells Vol. 21 ( 7 ) page： 696-705 2016.7

The chaperone complex BAG2-HSC70 regulates localization of C. elegans Leucine-rich repeat kinase LRK-1 in the Golgi Reviewed

Fukuzono T, Pastuhov SI, Fukushima O, Li C, Hattori A, Iemura S, Natsume T, Shibuya H, Hanafusa H, Matsumoto K, Hisamoto N.

Genes to Cells page： in press 2016

MAP kinase cascades regulating axon regeneration in C. elegans. Invited Reviewed

Pastuhov SI, Hisamoto N., Matsumoto K.

Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. Vol. 91 page： 63-75 2015

he Caenorhabditis elegans JNK Signaling Pathway Activates Expression of Stress Response Genes by Derepressing the Fos/HDAC Repressor Complex Reviewed

Hattori A, Mizuno T, Akamatsu M, Hisamoto N, Matsumoto K.

PLoS Genetics Vol. 9 page： e1003315 2013.2

Forgetting in C. elegans is accelerated by neuronal communication via the TIR-1/JNK-1 pathway. Reviewed

Inoue A, Sawatari E, Hisamoto N, Kitazono T, Teramoto T, Fujiwara M, Matsumoto K, Ishihara T.

Cell Reports Vol. 3 page： 808-816 2013.2

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

The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca(2+) responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting.

DOI： 10.1016/j.celrep.2013.02.019

Dysregulated LRRK2 signaling in response to endoplasmic reticulum stress leads to dopaminergic neuron degeneration in C. elegans. Reviewed

Yuan Y, Cao P, Smith MA, Kramp K, Huang Y, Hisamoto N, Matsumoto K, Hatzoglou M, Jin H, Feng Z.

PLoS One Vol. 6 ( 8 ) page： e22354 2011.8

Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways. Reviewed

Nix P, Hisamoto N, Matsumoto K, Bastiani M.

Proceedings of the National Academy of Sciences of the United States of America Vol. 108 ( 26 ) page： 10738-10743 2011.6

Regulation of Anoxic Death in Caenorhabditis elegans by Mammalian Apoptosis Signal-Regulating Kinase (ASK) Family Proteins. Reviewed

Hayakawa T, Kato K, Hayakawa R, Hisamoto N, Matsumoto K, Takeda K, Ichijo H.

Genetics Vol. 187 ( 3 ) page： 785-792 2011.3

The C. elegans JIP3 protein UNC-16 functions as an adaptor to link kinesin-1 with cytoplasmic dynein. Reviewed

Arimoto M, Koushika SP, Choudhary B, Li C, Matsumoto K, Hisamoto N.

Journal of Neuroscience Vol. 31 page： 2216-2224 2011.2

The ERK-MAPK pathway regulates longevity through SKN-1 and insulin-like signaling in Caenorhabditis elegans. Reviewed

Okuyama T, Inoue H, Ookuma S, Satoh T, Kano K, Honjoh S, Hisamoto N, Matsumoto K, Nishida E.

Journal of Biological Chemistry Vol. 285 ( 39 ) page： 30274-30281 2010.9

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

It has not been determined yet whether the ERK-MAPK pathway regulates longevity of metazoans. Here, we show that the Caenorhabditis elegans ERK cascade promotes longevity through the two longevity-promoting transcription factors, SKN-1 and DAF-16. We find that RNAi of three genes, which constitute the ERK cascade (lin-45/RAF1, mek-2/MEK1/2, and mpk-1/ERK1/2), results in reduction of life span. Moreover, RNAi of lip-1, the gene encoding a MAPK phosphatase that inactivates MPK-1, increases life span. Epistasis analyses show that the ERK (MPK-1) cascade-mediated life span extension requires SKN-1, whose function is mediated, at least partly, through DAF-2/DAF-16 insulin-like signaling. MPK-1 phosphorylates SKN-1 on the key sites that are required for SKN-1 nuclear accumulation. Our results also show that one mechanism by which SKN-1 regulates insulin-like signaling is through the regulation of expression of insulin-like peptides. Our findings thus identify a novel ERK-MAPK-mediated signaling pathway that promotes longevity.

Phosphorylation of the conserved transcription factor ATF-7 by PMK-1 p38 MAPK regulates innate immunity in Caenorhabditis elegans. Reviewed

Shivers RP, Pagano DJ, Kooistra T, Richardson CE, Reddy KC, Whitney JK, Kamanzi O, Matsumoto K, Hisamoto N, Kim DH

PLoS Genetics Vol. 6 ( 4 ) page： e1000892 2010.4

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

Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP-responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1-mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1-regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1-regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1-regulated genes observed in the pmk-1 null mutant, the induction of PMK-1-regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.

The Caenorhabditis elegans Ste20-related kinase and Rac-type small GTPaseregulate the JNK signaling pathway mediating the stress response. Reviewed

Fujiki K, Mizuno T, Hisamoto N, Matsumoto K.

Molecular and Cellular Biology Vol. 30 ( 4 ) page： 995-1003 2010.2

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

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a JNK-like MAPK signaling pathway, which is regulated by MLK-1 MAPKKK, MEK-1 MAPKK and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.

The germinal center kinase GCK-1 Is a negative regulator of MAP kinase activation and apoptosis in the C. elegans germline. Reviewed

Schouest KR, Kurasawa Y, Furuta T, Hisamoto N, Matsumoto K, Schumacher JM.

PLos One Vol. 4 ( 10 ) page： e7450 2009.10

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

The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts.

Caenorhabditis elegans FOS-1 and JUN-1 regulate plc-1 expression in the spermatheca to control ovulation. Reviewed

Hiatt SM, Duren HM, Shyu YJ, Ellis RE, Hisamoto N, Matsumoto K, Kariya K, Kerppola TK, Hu CD.

Molecular Biology of the Cell Vol. 20 ( 17 ) page： 3888-95 2009.9

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

Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.

LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans. Reviewed

Saha S, Guillily MD, Ferree A, Lanceta J, Chan D, Ghosh J, Hsu CH, Segal L, Raghavan K, Matsumoto K, Hisamoto N, Kuwahara T, Iwatsubo T, Moore L, Goldstein L, Cookson M, Wolozin B.

Journal of Neuroscience Vol. 29 ( 29 ) page： 9210-8 2009.7

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

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

*C. elegans WNK-STE20 pathway regulates tube formation by modulating ClC channel activity Reviewed

Naoki Hisamoto, Tetsuo Moriguchi, Seiichi Urushiyama, Shohei Mitani, Hiroshi Shibuya, Kunihiro Matsumoto

EMBO reports Vol. 9 page： 70-75 2008

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

WNK kinases comprise a small group of unique serine/threonine protein kinases conserved among multicellular organisms. Mutations in WNK1/4 cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to the STE20 kinases and ion carriers, but the underlying molecular mechanisms by which WNKs regulate cellular processes in whole animals are unknown. The C. elegans WNK-like kinase WNK-1 interacts with and phosphorylates GCK-3, a STE20-like kinase that is known to inactivate CLH-3, a CIC chloride channel. The wnk-1 or gck-3 deletion mutation causes an Exc phenotype, a defect in the tubular extension of excretory canals. Expression of the activated form of GCK-3 or the clh-3 deletion mutation can partially suppress wnk-1 or gck-3 defects, respectively. These results suggest that WNK-1 controls the tubular formation of excretory canals by activating GCK-3, resulting in downregulation of CIC channel activity.

Role of the C. elegans Shc adaptor protein in the JNK signaling pathway. Reviewed

Mizuno T, Fujiki K, Sasakawa A, Hisamoto N, Matsumoto K.

Molecular and Cellular Biology Vol. in press 2008

*LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins Reviewed

Sakaguchi-Nakashima A, Meir JY, Jin Y, Matsumoto K, Hisamoto N.

Current biology Vol. 17 ( 7 ) page： 592-598 2007.4

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

Neurons are polarized cells that contain distinct sets of proteins in their axons and dendrites. Synaptic vesicles (SV) and many SV proteins are exclusively localized in the presynaptic regions but not in dendrites. Despite their fundamental importance, the mechanisms underlying the polarized localization of SV proteins remain unclear. The transparent nematode Caenorhabditis elegans can be used to examine sorting and transport of SV proteins in vivo. Here, we identify a novel protein kinase LRK-1, a C. elegans homolog of the familial Parkinsonism gene PARK8/LRRK2 that is required for polarized localization of SV proteins. In lrk-1 deletion mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons. This aberrant localization of SV proteins in the dendrites is dependent on the AP-1 mu1 clathrin adaptor UNC-101, which is involved in polarized dendritic transport, but not on UNC-104 kinesin, which is required for the transport of SV to presynaptic regions. The LRK-1 proteins are localized in the Golgi apparatus. These results suggest that the LRK-1 protein kinase determines polarized sorting of SV proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi.

?WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1. Reviewed

Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume T, Matsumoto K, Shibuya H.

Journal of Biological Chemistry Vol. 280 ( 52 ) page： 42685-42693 2005.12

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

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.

Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Reviewed

An JH, Vranas K, Lucke M, Inoue H, Hisamoto N, Matsumoto K, Blackwell TK.

Proceedings of the National Academy of Sciences of the United States of America Vol. 102 ( 45 ) page： 16275-16280 2005.11

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

Oxidative stress plays a central role in many human diseases and in aging. In Caenorhabditis elegans the SKN-1 protein induces phase II detoxification gene transcription, a conserved oxidative stress response, and is required for oxidative stress resistance and longevity. Oxidative stress induces SKN-1 to accumulate in intestinal nuclei, depending on p38 mitogen-activated protein kinase signaling. Here we show that, in the absence of stress, phosphorylation by glycogen synthase kinase-3 (GSK-3) prevents SKN-1 from accumulating in nuclei and functioning constitutively in the intestine. GSK-3 sites are conserved in mammalian SKN-1 orthologs, indicating that this level of regulation may be conserved. If inhibition by GSK-3 is blocked, background levels of p38 signaling are still required for SKN-1 function. WT and constitutively nuclear SKN-1 comparably rescue the skn-1 oxidative stress sensitivity, suggesting that an inducible phase II response may provide optimal stress protection. We conclude that (i) GSK-3 inhibits SKN-1 activity in the intestine, (ii) the phase II response integrates multiple regulatory signals, and (iii), by inhibiting this response, GSK-3 may influence redox conditions.

?The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Reviewed

Inoue H, Hisamoto N, An JH, Oliveira RP, Nishida E, Blackwell TK, Matsumoto K.

Genes and Development Vol. 19 ( 19 ) page： 2278-2283 2005.10

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

The evolutionarily conserved p38 mitogen-activated protein kinase (MAPK) cascade is an integral part of the response to a variety of environmental stresses. Here we show that the Caenorhabditis elegans PMK-1 p38 MAPK pathway regulates the oxidative stress response via the CNC transcription factor SKN-1. In response to oxidative stress, PMK-1 phosphorylates SKN-1, leading to its accumulation in intestine nuclei, where SKN-1 activates transcription of gcs-1, a phase II detoxification enzyme gene. These results delineate the C. elegans p38 MAPK signaling pathway leading to the nucleus that responds to oxidative stress.

The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization. Reviewed

Sakamoto R, Byrd DT, Brown HM, Hisamoto N, Matsumoto K, Jin Y.

Molecular Biology of the Cell Vol. 16 ( 2 ) page： 483-496 2005.2

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

Kinesin-1 is a heterotetramer composed of kinesin heavy chain (KHC) and kinesin light chain (KLC). The Caenorhabditis elegans genome has a single KHC, encoded by the unc-116 gene, and two KLCs, encoded by the klc-1 and klc-2 genes. We show here that UNC-116/KHC and KLC-2 form a complex orthologous to conventional kinesin-1. KLC-2 also binds UNC-16, the C. elegans JIP3/JSAP1 JNK-signaling scaffold protein, and the UNC-14 RUN domain protein. The localization of UNC-16 and UNC-14 depends on kinesin-1 (UNC-116 and KLC-2). Furthermore, mutations in unc-16, klc-2, unc-116, and unc-14 all alter the localization of cargos containing synaptic vesicle markers. Double mutant analysis is consistent with these four genes functioning in the same pathway. Our data support a model whereby UNC-16 and UNC-14 function together as kinesin-1 cargos and regulators for the transport or localization of synaptic vesicle components.

Integration of Caenorhabditis elegans MAPK pathways mediating immunity and stress resistance by MEK-1 MAPK kinase and VHP-1 MAPK phosphatase. Reviewed

Kim DH, Liberati NT, Mizuno T, Inoue H, Hisamoto N, Matsumoto K, Ausubel FM.

Proceedings of the National Academy of Sciences of the United States of America Vol. 101 ( 30 ) page： 10990-10994 2004.7

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

The p38 and JNK classes of mitogen-activated protein kinases (MAPKs) have evolutionarily conserved roles in the control of cellular responses to microbial and abiotic stresses. The mechanisms by which crosstalk between distinct p38 and c-Jun N-terminal kinase (JNK) MAPK pathways occurs with resultant integration of signaling information have been difficult to establish, particularly in the context of whole organism physiology. In Caenorhabditis elegans a PMK-1 p38 MAPK pathway is required for resistance to bacterial infection, and a KGB-1 JNK-like MAPK pathway has recently been shown to mediate resistance to heavy metal stress. Here, we show that two components of the KGB-1 pathway, MEK-1 MAPK kinase (MAPKK), a homolog of mammalian MKK7, and VHP-1 MAPK phosphatase (MKP), a homolog of mammalian MKP7, also regulate pathogen resistance through the modulation of PMK-1 activity. The regulation of p38 and JNK-like MAPK pathways mediating immunity and heavy metal stress by common MAPKK and MKP signaling components suggests pivotal roles for MEK-1 and VHP-1 in the integration of diverse stress signals contributing to pathogen resistance in C. elegans. In addition, these data point to mechanisms in multicellular organisms by which signals transduced by distinct MAPK pathways may be subject to physiological integration at the level of regulation of MAPK activity by MAPKKs and MKPs.

Roles of MAP kinase cascades in Caenorhabditis elegans. Invited

Sakaguchi A, Matsumoto K, Hisamoto N.

Journal of Biochemistry (Tokyo) Vol. 136 ( 1 ) page： 7-11 2004.7

Authorship：Lead author Language：English

Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that are activated by diverse stimuli such as growth factors, cytokines, neurotransmitters and various cellular stresses. MAPK cascades are generally present as three-component modules, consisting of MAPKKK, MAPKK and MAPK. The precise molecular mechanisms by which these MAPK cascades transmit signals is an area of intense research, and our evolving understanding of these signal cascades has been facilitated in great part by genetic analyses in model organisms. One organism that has been commonly used for genetic manipulation and physiological characterization is the nematode Caenorhabditis elegans. Genes sequenced in the C. elegans genome project have furthered the identification of components involved in several MAPK pathways. Genetic and biochemical studies on these components have shed light on the physiological roles of MAPK cascades in the control of cell fate decision, neuronal function and immunity in C. elegans.

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response. Reviewed

Mizuno T, Hisamoto N, Terada T, Kondo T, Adachi M, Nishida E, Kim DH, Ausubel FM, Matsumoto K.

EMBO Journal Vol. 23 ( 11 ) page： 2226-2234 2004.6

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

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.

An NDPase links ADAM protease glycosylation with organ morphogenesis in C. elegans. Reviewed

Nishiwaki K, Kubota Y, Chigira Y, Roy SK, Suzuki M, Schvarzstein M, Jigami Y, Hisamoto N, Matsumoto K.

Nature Cell biology Vol. 6 ( 1 ) page： 31-37 2004.1

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

In the nematode Caenorhabditis elegans, the gonad acquires two U-shaped arms through the directed migration of its distal tip cells (DTCs), which are located at the tip of the growing gonad arms. A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, regulates directional migration of DTCs: MIG-17 is synthesized and secreted from the muscle cells of the body wall, and diffuses to the gonad where it is required for DTC migration. The mig-23 mutation causes defective migration of DTCs and interacts genetically with mig-17. Here, we report that mig-23 encodes a membrane-bound nucleoside diphosphatase (NDPase) required for glycosylation and proper localization of MIG-17. Our findings indicate that an NDPase affects organ morphogenesis through glycosylation of the MIG-17 ADAM protease.

SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis elegans. Reviewed

EMBO Reports Vol. 3 ( 1 ) page： 56-62 2002

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

The mitogen-activated protein kinase (MAPK) pathway is a highly conserved signaling cascade that converts extracellular signals into various outputs. In Caenorhabditis elegans, asymmetric expression of the candidate odorant receptor STR-2 in either the left or the right of two bilaterally symmetrical olfactory AWC neurons is regulated by axon contact and Ca2+ signaling. We show that the MAPK kinase (MAPKK) SEK-1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK-1 functions in a pathway downstream of UNC-43 and NSY-1, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and MAPK kinase kinase (MAPKKK), respectively. Thus, the NSY-1-SEK-1-MAPK cascade is activated by Ca2+ signaling through CaMKII and establishes asymmetric cell fate decision during neuronal development.

A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Reviewed

Science Vol. 297 ( 5581 ) page： 623-626 2002

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

A genetic screen for Caenorhabditis elegans mutants with enhanced susceptibility to killing by Pseudomonas aeruginosa led to the identification of two genes required for pathogen resistance: sek-1, which encodes a mitogen-activated protein (MAP) kinase kinase, and nsy-1, which encodes a MAP kinase kinase kinase. RNA interference assays and biochemical analysis established that a p38 ortholog, pmk-1, functions as the downstream MAP kinase required for pathogen defense. These data suggest that this MAP kinase signaling cassette represents an ancient feature of innate immune responses in evolutionarily diverse species.

*The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates. Reviewed

Cell Vol. 105 ( 2 ) page： (221-232) 2001

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

A stochastic cell fate decision mediated by axon contact and calcium signaling causes one of the two bilaterally symmetric AWC neurons, either AWCL or AWCR, to express the candidate olfactory receptor str-2. nsy-1 mutants express str-2 in both neurons, disrupting AWC asymmetry. nsy-1 encodes a homolog of the human MAP kinase kinase kinase (MAPKKK) ASK1, an activator of JNK and p38 kinases. Based on genetic epistasis analysis, nsy-1 appears to act downstream of the CaMKII unc-43, and NSY-1 associates with UNC-43, suggesting that UNC-43/CaMKII activates the NSY-1 MAP kinase cassette. Mosaic analysis demonstrates that UNC-43 and NSY-1 act primarily in a cell-autonomous execution step that represses str-2 expression in one AWC cell, downstream of the initial lateral signaling pathway that coordinates the fates of the two cells.

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK. Reviewed

Journal of Biological Chemistry Vol. 276 ( 26 ) page： 24396-24400 2001

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

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.

UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans. Reviewed

Neuron Vol. 32 ( 5 ) page： 787-800 2001

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

Transport of synaptic components is a regulated process. Loss-of-function mutations in the C. elegans unc-16 gene result in the mislocalization of synaptic vesicle and glutamate receptor markers. unc-16 encodes a homolog of mouse JSAP1/JIP3 and Drosophila Sunday Driver. Like JSAP1/JIP3, UNC-16 physically interacts with JNK and JNK kinases. Deletion mutations in Caenorhabditis elegans JNK and JNK kinases result in similar mislocalization of synaptic vesicle markers and enhance weak unc-16 mutant phenotypes. unc-116 kinesin heavy chain mutants also mislocalize synaptic vesicle markers, as well as a functional UNC-16::GFP. Intriguingly, unc-16 mutations partially suppress the vesicle retention defect in unc-104 KIF1A kinesin mutants. Our results suggest that UNC-16 may regulate the localization of vesicular cargo by integrating JNK signaling and kinesin-1 transport.

A putative GDP-GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans. Reviewed

EMBO Reports Vol. 2 ( 6 ) page： 530-535 2001

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

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.

*A metalloprotease disintegrin that controls cell migration in Caenorhabditis elegans. Reviewed

Science Vol. 288 ( 5474 ) page： 2205-2208 2000

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

In Caenorhabditis elegans, the gonad acquires two U-shaped arms by the directed migration of its distal tip cells (DTCs) along the body wall basement membranes. Correct migration of DTCs requires the mig-17 gene, which encodes a member of the metalloprotease-disintegrin protein family. The MIG-17 protein is secreted from muscle cells of the body wall and localizes in the basement membranes of gonad. This localization is dependent on the disintegrin-like domain of MIG-17 and its catalytic activity. These results suggest that the MIG-17 metalloprotease directs migration of DTCs by remodeling the basement membrane.

SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development Reviewed

Mechanisms of Development Vol. 99 ( 1-2 ) page： 51-64 2000

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

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.

MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans. Reviewed

Nature Vol. 399 ( 6738 ) page： 793-797 1999

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

The signalling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. Here we show that the genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. We conclude that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, as TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1.

Molecular cloning and expression of a stress-responsive mitogen-activated protein kinase-related kinase from Tetrahymena cells. Reviewed

Journal of Biological Chemistry Vol. 274 ( 15 ) page： 9976-83 1999

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

To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stress-activated protein kinase which are known to be involved in various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15 degrees C (0.8 degrees C/min). Interestingly, osmotic shock either by sorbitol (100-200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells.

*A Caenorhabditis elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons. Reviewed

EMBO J. Vol. 18 ( 13 ) page： 3604-3615 1999

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

The c-Jun N-terminal kinase (JNK) of the MAP kinase superfamily is activated in response to a variety of cellular stresses and is involved in apoptosis in neurons. However, the roles of the JNK signaling pathway in the nervous system are unknown. The genes for the Caenorhabditis elegans homolog of JNK, JNK-1, and its direct activator, JKK-1, were isolated based on their abilities to function in the Hog1 MAP kinase pathway in yeast. JKK-1 is a member of the MAP kinase kinase superfamily and functions as a specific activator of JNK. Both jnk-1 and jkk-1 are expressed in most neurons. jkk-1 null mutant animals exhibit defects in locomotion that can be rescued by the conditional expression of JKK-1 in mutant adults, suggesting that the defect is not due to a developmental error. Furthermore, ectopic expression of JKK-1 in type-D motor neurons is sufficient to rescue the movement defect. Thus, the C.elegans JNK pathway functions in type-D GABAergic motor neurons and thereby modulates coordinated locomotion.

The EGP1 gene may be a positive regulator of protein phosphatase type 1 in the growth control of Saccharomyces cerevisiae. Reviewed

Molecular and Cellular Biology Vol. 15 ( 7 ) page： 3767-3776 1995

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

The Saccharomyces cerevisiae GLC7 gene encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is required for cell growth. A cold-sensitive glc7 mutant (glc7Y170) arrests in G2/M but remains viable at the restrictive temperature. In an effort to identify additional gene products that function in concert with PP1 to regulate growth, we isolated a mutation (gpp1) that exacerbated the growth phenotype of the glc7Y170 mutation, resulting in rapid death of the double mutant at the nonpermissive temperature. We identified an additional gene, EGP1, as an extra-copy suppressor of the glc7Y170 gpp1-1 double mutant. The nucleotide sequence of EGP1 predicts a leucine-rich repeat protein that is similar to Sds22, a protein from the fission yeast Schizosaccharomyces pombe that positively modulates PP1. EGP1 is essential for cell growth but becomes dispensable upon overexpression of the GLC7 gene. Egp1 and PP1 directly interact, as assayed by coimmunoprecipitation. These results suggest that Egp1 functions as a positive modulator of PP1 in the growth control of S. cerevisiae.

The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M. Reviewed

Molecular and cellular biology Vol. 14 ( 5 ) page： 3158-65 1995

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

We isolated a mutant carrying a conditional mutation in the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by selection of suppressors that restored the growth defect of cdc24 mutants at high temperature and simultaneously conferred cold-sensitive growth. This cold sensitivity for growth is caused by a single mutation (glc7Y-170) at position 170 of the Glc7 protein, resulting in replacement of cysteine with tyrosine. Genetic analysis suggested that the glc7Y-170 allele is associated with a recessive negative phenotype, reducing the activity of Glc7 in the cell. The glc7Y-170 mutant missegregated chromosome III at the permissive temperature, arrested growth as large-budded cells at the restrictive temperature, exhibited a significant increase in the number of nuclei at or in the neck, and had a short spindle. Furthermore, the glc7Y-170 mutant exhibited a high level of CDC28-dependent protein kinase activity when incubated at the restrictive temperature. These findings suggest that the glc7Y-170 mutation is defective in the G2/M phase of the cell cycle. Thus, type 1 protein phosphatase in Saccharomyces cerevisiae is essential for the G2/M transition.

DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication. Reviewed

Seino H, Hisamoto N, Uzawa S, Sekiguchi T, Nishimoto T.

Journal of Cell Science Vol. 102 ( 3 ) page： 393-400 1992

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

The RCC1 protein that is required for coupling mitosis with the S phase has a DNA-binding domain in the N-terminal region outside the repeat. We found that RCC1 protein without any DNA-binding activity complemented the tsBN2 mutation with the same efficiency as that of intact RCC1 protein. In ts+ transformants of tsBN2 cells transfected with the RCC1 cDNA lacking the DNA-binding domain, an endogenous RCC1 disappeared at 39.5 degrees C, and the deleted RCC1 protein encoded by the transfected cDNA was found in the cytoplasm, but a significant amount of it was also found in the nuclei. This deleted RCC1 protein was eluted from the nuclei with the same concentration of NaCl and DNase I as was used for the intact RCC1 protein in BHK21 cells. Furthermore, the deleted RCC1 protein co-migrated with the nucleosome fraction on sucrose density gradient analysis. These results indicate that the RCC1 protein binds chromatin with the aid of other unknown protein(s). Thus, the DNA-binding domain of RCC1 protein is not essential for coupling between the S and M phases, but was shown instead to function as a nuclear translocation signal.

RCCl is a nuclear protein required for coupling activation of cdc2 kinase with DNA synthesis and for start of the cell cycle.

Seino H, Nishitani H, Seki T, Hisamoto N, Tazunoki T, Shiraki N, Ohtsubo M, Yamashita K, Sekiguchi T, Nishimoto T.

Cold Spring Harbor Symposium of Quantitative Biology Vol. 56 page： 367-375 1991

The human CCG1 gene, essential for progression of the G1 phase, encodes a 210-kilodalton nuclear DNA-binding protein. Reviewed

Sekiguchi T, Nohiro Y, Nakamura Y, Hisamoto N, Nishimoto T

Molecular and Cellular Biology Vol. 11 ( 6 ) page： 3317-3325 1991

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

The human CCG1 gene complements tsBN462, a temperature-sensitive G1 mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCG1 cDNA. The newly cloned CCG1 cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCG1 protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase II and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer. CCG1 contains a sequence similar to the putative DNA-binding domain of HMG1 in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCG1 was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.

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