Fukuzono, Takashi et al. (2011) International Worm Meeting "WNK-1 negatively regulates formation of diacylglycerol in C. elegans."

Matsumoto, Kunihiro, Hisamoto, Naoki, Fukuzono, Takashi

[International Worm Meeting, 2011]

WNK kinases are the causative genes of pseudohypoaldosteronism type II (PHA-II) disease, which is a familial autosomal-dominant illness featuring salt-dependent hypertension with hyperkalemia in human. We have previously shown that wnk-1, a WNK homolog in C. elegans, regulates excretory canal morphology and larval growth in worms. To identify players acting with WNK-1, we screened suppressor mutants for the larval arrest phenotype associated with the wnk-1 deletion mutation. One of the suppressors, sowl (suppressor of wnk-1 lethality)-1 exhibits partial embryonic lethality (Emb) and polarity defect (Par) in the two-cell stage embryo. The sowl-1 gene encodes an acylglycerol O-acyltransferase, which is required for triacylglycerol (TAG) synthesis. The TAG synthesis is important for storage and delivery of fatty acids, raising the possibility that the phenotypes observed in sowl-1 mutants are caused by the defect in fatty acid metabolism. In fact, we found that addition of TAG or fatty acids was able to rescue the Par phenotype in sowl-1 mutants. Furthermore, a diacylglycerol (DAG) analog PMA efficiently rescued both Emb and Par phenotypes in sowl-1 mutants. On the other hand, addition of excess PMA to wild-type animals induced Larval arrest and the defect in the extension of excretory canals, whose phenotypes are similar to those observed in wnk-1 mutants. These results suggest that WNK-1 negatively regulates DAG formation in C. elegans.

Kitamura S et al. (1994) Worm Breeder's Gazette "The Troponin C Gene, tnc-1, of Caenorhabditis elegans: Genome Structure and Mutation Sites of pat-10"

Sakube Y, Kagawa H, Williams BD, Matsumoto S, Kitamura S

[Worm Breeder's Gazette, 1994]

The troponin C gene, tnc-1 of Caenorhabditis elegans: Genome structure and mutation sites of pat-10 S. KITAMURA, B. WILLIAMS*, Y. SAKUBE, S. MATSUMOTO and H. KAGAWA, Dept. of Biol., Fac. of Sci., Okayama University, Japan, 700. *;Dept. of Gen., Washington Univ. School of Med., St. Louis

Fujiki, Kota et al. (2009) International Worm Meeting "C. elegans MLK-1 MAPKKK is regulated by double phosphorylation in JNK-mediated stress response pathway."

Matsumoto, Kunihiro, Fujiki, Kota, Mizuno, Tomoaki, Hisamoto, Naoki

[International Worm Meeting, 2009]

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 C. elegans, the stress response is controlled by a JNK-like MAPK signaling pathway composed of MLK-1 MAPKKK, MEK-1 MAPKK and KGB-1 JNK-like MAPK. As components functioning in the KGB-1 pathway, we identified the max-2 and shc-1 genes, which encode C. elegans homologs of PAK-like kinase and Shc, respectively. Biochemical analysis revealed that MAX-2 phosphorylates MLK-1 at Ser-355 in the T-loop of the MLK-1 kinase domain, resulting in its activation. We also found that phosphorylation of MLK-1 at Tyr-940 creates a binding site for SHC-1 and results in the recruitment of the SHC-1-MEK-1 complex to MLK-1. These results suggest that MLK-1 is regulated by double phosphorylation: 1) phosphorylation of Ser-355 regulates its kinase activity; and 2) phosphorylation of Tyr-940 regulates the association with SHC-1, consequently interacting with its substrate, MEK-1. Thus, regulation of MLK-1 by the double phosphorylation in the KGB-1 pathway can modulate the amplitude and duration of the signal in the stress response.

Pastuhov, Strahil Iv et al. (2013) International Worm Meeting "Endocannabinoid AEA as injury signal in axon regeneration."

Hisamoto, Naoki, Matsumoto, Kunihiro, Pastuhov, Strahil Iv

[International Worm Meeting, 2013]

The endocannabinoid arachidonoyl ethanolamide (AEA) inhibits axon regeneration of D-type motor neurons in Caenorhabditis elegans by regulating the activity of the JNK MAP kinase cascade (Pastuhov SI et al (2012), Nat Commun. 3:1136). AEA levels are controlled through enzymatic synthesis and degradation. Our previous work demonstrated that inactivation of the AEA degradation pathway inhibits axon regeneration. For example, deletion mutation in the gene for fatty acid amide hydrolase (FAAH), the enzyme that inactivates AEA by hydrolysis, causes defect in axon regeneration due to accumulation of high levels of endogenous AEA. This time, we focused on the synthesis of AEA in axon regeneration. NAPE-1 is a worm homologue of NAPE-PLD, an enzyme involved in AEA synthesis. We found that the nape-1 null mutation suppressed the faah-1 defect in axon regeneration. Conversely, overexpression of nape-1 in D-type motor neurons by the unc-25 promoter inhibited axon regeneration after laser surgery. These results suggest that the inhibition of axon regeneration by NAPE-1 occurs at restricted area around the injured neuron. Interestingly, when nape-1 was overexpressed in touch receptor neurons by the mec-7 promoter, D-type motor neurons were able to regenerate after laser surgery, but their regenerating axons tended to make a sharp turn upon approaching the lateral midline, where axons of PLM touch neurons extend. These results suggest that high local levels of AEA inhibit axon regeneration and that a gradient of lower concentration affects the guidance of the regenerating axon.

Alam, Tanimul et al. (2015) International Worm Meeting "A tensin3 homolog SVH-6 regulates axon regeneration by stabilization of a MET-like receptor tyrosine kinase SVH-2."

Asai, Kazuma, Matsumoto, Kunihiro, Alam, Tanimul, Hisamoto, Naoki

[International Worm Meeting, 2015]

The Jun N-terminal kinase (JNK) MAP kinase (MAPK) pathway, which is composed of MLK-1 (MAPKKK), MEK-1 (MAPKK) and KGB-1 (MAPK), plays a pivotal role in axon regeneration in C. elegans. Recently, we have identified an HGF-like growth factor SVH-1 and a Met-like receptor tyrosine kinase SVH-2 as positive regulators acting upstream of JNK MAPK pathway in axon regeneration. However, other molecular factors that regulate this pathway are not well understood. In this study, we identified the svh-6 gene, which encodes a homolog of mammalian tensin3, as a regulator of axon regeneration. In svh-6 mutants, frequency of axon regeneration was significantly reduced. This defect was rescued by the expression of the svh-6 gene under the D-type motor neuron specific promoter, suggesting that SVH-6 acts cell-autonomously. SVH-6 contains an Actin Binding Domain (ABD), a Src Homology 2 (SH2) domain, and a Phospho-Tyrosine Binding (PTB) domain. We found that both SH2 and PTB domains, but not ABD, are required for SVH-6 function in axon regeneration. Further genetic analysis revealed that SVH-6 acts upstream of SVH-2 in axon regeneration. Biochemical analysis using mammalian cultured cells showed that SVH-6 lacking ABD (SVH-6 delta-ABD) associates with Tpr-SVH-2C, an activated form of cytoplasmic domain of SVH-2. This interaction is dependent on both the kinase activity of Tpr-SVH-2C and the SH2 domain of SVH-6, indicating that the SH2 domain of SVH-6 can interact with the activated form of SVH-2 cytoplasmic domain. To evaluate the in vivo function of SVH-6, we overexpressed SVH-6 delta-ABD and determined the effect on the SVH-2 protein in severed axons. In wild-type animals, the SVH-2 protein fused to VENUS fluorescent protein (SVH-2::VENUS) is unstable and not visible by fluorescent microscope in both unsevered and severed neurons. However, SVH-6 delta-ABD overexpression enabled it to be visible at proximal retraction bulb in severed neurons. Thus, these results suggest that SVH-6 interacts with SVH-2 via SH2 domain and regulates axon regeneration by stabilizing the active SVH-2 protein at retraction bulb.

Hattori, Ayuna et al. (2011) International Worm Meeting "The KGB-1 JNK signaling pathway negatively regulates FOS-1 transcription factor in stress response."

Mizuno, Tomoaki, Matsumoto, Kunihiro, Hattori, Ayuna, Hisamoto, Naoki

[International Worm Meeting, 2011]

The JNK MAP kinase (MAPK) pathway plays a pivotal role in the various stress responses of evolutionarily diverse species. In C. elegans, a JNK-like MAPK pathway composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK controls stress response. Previously, we found that KGB-1 negatively regulates FOS-1, a bZIP transcription factor homologous to human Fos, through its phosphorylation of FOS-1 in heavy metal stress response. However, a downstream target of FOS-1 has not been identified. To identify genes whose expression is regulated by the KGB-1 pathway, we executed DNA microarray analysis and the results were confirmed by quantitative PCR. We identified three kreg (KGB-1 regulated gene) genes, kreg-1~3, whose expression was induced by copper in a KGB-1-dependent manner. Furthermore, their inductions were increased in fos-1 knockdown animals. Knockdown of kreg-1~3 caused a partial sensitivity to heavy metals. Taken together, these data suggest that KGB-1 induces expression of genes including kreg-1~3 by negatively regulating FOS-1 in heavy metal stress response.

Hattori, Ayuna et al. (2009) International Worm Meeting "KGB-1, a JNK-like MAPK, negatively regulates FOS-1 transcription factor in stress response."

Hisamoto, Naoki, Matsumoto, Kunihiro, Hattori, Ayuna, Mizuno, Tomoaki

[International Worm Meeting, 2009]

The JNK MAP kinase (MAPK) pathway plays a pivotal role in the various stress responses in evolutionarily diverse species. In C. elegans, a JNK-like MAPK pathway composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK controls stress response. However, other components functioning together with this pathway have not been well characterized. Recently, we identified cdk-8 gene, encoding CDK8 module subunits of Mediator complex, as a novel factor functioning together with the KGB-1 pathway. The cdk-8 mutation caused a partial sensitivity to heavy metal stress and enhanced the stress sensitivity of kgb-1 mutants. These results suggest that CDK-8 regulates the stress response in parallel to the KGB-1 pathway. The fact that JNK phosphorylates transcription factors raise the possibility that KGB-1 also regulates transcription factors. To identify the novel downstream factors regulated by KGB-1, we performed yeast two-hybrid screening using KGB-1 as a bait. As a result, we isolated FOS-1, a bZIP transcription factor homologous to human Fos, as a KGB-1 binding protein. Further biochemical analysis showed that FOS-1 is phosphorylated by KGB-1. In addition, the knock down of FOS-1 by RNAi suppressed the stress sensitivity of kgb-1 mutants. These results suggest that KGB-1 negatively regulates FOS-1 through phosphorylation of FOS-1.

Hisamoto, Naoki et al. (2013) International Worm Meeting "The C. elegans plasminogen/HGF-like protein SVH-1 is required for larval developmental growth."

Li, Chun, Matsumoto, Kunihiro, Hisamoto, Naoki, Yoshida, Motoki

[International Worm Meeting, 2013]

The plasminogen family consists of both the active proteases including plasminogen and the protease-inactive growth factors including hepatocyte growth factor (HGF). C. elegans plasminogen/HGF-like protein SVH-1 acts as a growth factor-like molecule that regulates axon regeneration via an HGF receptor-like protein SVH-2 and KGB-1 MAP kinase cascade (Li et al., Nature Neurosci. 15, p551, 2012). Interestingly, we found that the svh-1 null mutation caused the larval lethal phenotype, which was not observed in svh-2 or kgb-1 null mutants. The svh-1 null mutant worms arrested at L2 stage with the starved appearance, suggesting that the mutant worms have a defect in taking foods. The feeding assay using the fluorescent beads and the observation of the pharyngeal pumping revealed that svh-1 mutant worms exhibited the progressive feeding defect associated with abnormal pharyngeal contraction. Rescue experiments showed that protease activity was essential for larval growth but not for axon regeneration. These results suggest that SVH-1 is involved in the regulation of pharyngeal contraction via its protease activity.

Alam, Tanimul et al. (2013) International Worm Meeting "CED-10 Rac signaling pathway regulates axon regeneration via JNK MAPK pathway."

Alam, Tanimul, Hisamoto, Naoki, Matsumoto, Kunihiro, Hirose, Kazuya, Pastuhov, Strahil

[International Worm Meeting, 2013]

Axon regeneration is regulated by JNK MAPK pathway, which is conserved among a wide variety of organisms. The C.elegans JNK MAPK pathway is composed of MLK-1 (MAPKKK), MEK-1 (MAPKK) and KGB-1 (MAPK). We have previously identified MAX-2, a mammalian PAK-like kinase, and MIG-2 GTPase as upstream activators of the JNK pathway in heavy metal stress response (Fujiki et al., MCB 30, p995, 2010). We found that max-2 mutant worms exhibited axon regeneration defect. However, the mig-2 mutation had no effect on axon regeneration. We found that MAX-2 associated with GTP-bound CED-10, a Rac-type GTPase, and that the ced-10 mutation was defective in axon regeneration. Furthermore, we found that CED-5, a guanine nucleotide exchange factor of CED-10, was also required for efficient axon regeneration. These results suggest that a Rac signaling pathway, composed of CED-5, CED-10 and MAX-2, acts as one of the upstream activators of JNK pathway in axon regeneration.

Li, Chun et al. (2011) International Worm Meeting "Genome-wide RNAi screening for vhp-1 suppressors in C. elegans."

Mizuno, Tomoaki, Li, Chun, Bastiani, Michael, Nix, Paola, Hisamoto, Naoki, Kanao, Shuka, Matsumoto, Kunihiro

[International Worm Meeting, 2011]

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 p38 and JNK MAPK pathways, consisting of DLK-1 (MAPKKK)/MKK-4 (MAPKK)/PMK-3 (MAPK) and MLK-1(MAPKKK)/MEK-1(MAPKK)/KGB-1 (MAPK), respectively. The vhp-1 gene encodes an MAPK phosphatase that negatively regulates both MAPK pathways. Animals with the vhp-1 deletion are arrested during larval development due to hyperactivation of the MAPK pathways. To identify components involved in MAPK-mediated signaling, we performed a genome-wide RNA interference (RNAi) screening for suppressors of the vhp-1 lethality. With this RNAi screening, we isolated dlk-1, pmk-3, cebp-1, mlk-1, mek-1 and kgb-1 as vhp-1 suppressors. Furthermore, we identified 10 novel svh (suppressor of vhp-1) genes. They encode protein kinases, transcription factors, mediator complexes, a receptor-like transmembrane protein and an RNA-binding protein. Here, we present results showing that a novel receptor-like transmembrane protein is required for neuronal regeneration in C. elegans.