avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.     

A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses

The c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was shown to be involved in the response to various stresses in cultured cells. However, there is little in vivo evidence indicating a role for a JNK pathway in the stress response of an organism. We identified the Caenorhabditis elegans mek-1 gene, which encodes a 347 amino acid protein highly homologous to mammalian MKK7, an activator of JNK. Mek-1 reporter fusion proteins are expressed in pharyngeal muscle, uterus, a portion of intestine, and neurons. A mek-1 deletion mutant is hypersensitive to copper and cadmium ions and to starvation. A wild-type mek-1 transgene rescued the hypersensitivity to the metal ions. Double mutants of mek-1 with an eat-5, eat-11 or eat-18 mutation, which are characterized by a limited feeding defect, showed distinct growth defects under normal conditions. Expression of an activated form of MEK-1 in the whole animal or specifically in the pharynx inhibited pharyngeal pumping. These results suggest a role for mek-1 in stress responses, with a focus in the pharynx and/or intestine.     

Distinct roles of the Src family kinases,SRC-1 and KIN-22, that are negatively regulated by CSK-1 in C. elegans

To elucidate the primitive roles of the Src family kinases (SFKs), here we characterized Caenorhabditis elegans orthologues of SFKs (src-1 and kin-22) and their regulator kinase Csk (csk-1). SRC-1 and KIN-22 possess the C-terminal regulatory tyrosines characteristic of SFKs, and their activities are negatively regulated by CSK-1 in a yeast expression system. The src-1 and csk-1 genes are co-expressed in some head neurons, the anchor cell and the tail region, while kin-22 and csk-1 genes are co-expressed in pharyngeal muscles and tail region. Expression of KIN-22 induced morphological defects in the pharynx, whereas expression of SRC-1 did not show any overt phenotype in adult. RNA interference of src-1, but not that of kin-22, caused a developmental arrest in early development. These results suggest that SRC-1 and KIN-22 play distinct roles under the control of CSK-1.     

PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response

In most animals, multiple genes encode protein kinase C (PKC) proteins. Pharmacological studies have revealed numerous roles for this protein family, yet the in vivo roles of specific PKC proteins and the functional targets of PKC activation are poorly understood. We find that in Caenorhabditis elegans, two PKC genes, pkc-1 and tpa-1, are required for mechanosensory response; the role of the nPKCε/η ortholog, pkc-1, was examined in detail. pkc-1 function is required for response to nose touch in adult C. elegans and pkc-1 likely acts in the interneurons that regulate locomotion which are direct synaptic targets of mechanosensory neurons. Previous studies have suggested numerous possible targets of pkc-1; our analysis indicates that pkc-1 may act via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. We find that ERK/MAPK pathway function is required for mechanosensory response in C. elegans and that at least one component of this pathway, lin-45 Raf, acts in interneurons of the mechanosensory circuit. Genetic analysis indicates that lin-45 and pkc-1 act together to regulate nose touch response. Thus, these results functionally link two conserved signaling pathways in adult C. elegans neurons and define distinct roles for PKC genes in vivo.     

Crystal structure of the MAP3K TAO2 kinase domain bound by an inhibitor staurosporine

Mitogen-activated protein kinase (MAPK) signal transduction pathways are ubiquitous ineukaryotic cells,which transfer signals from the cell surface to the nucleus,controlling multiple cellularprograms.MAPKs are activated by MAPK kinases [MAP2Ks or MAP/extracellular signal-regulated kinase(ERK) kinases (MEK)],which in turn are activated by MAPK kinase kinases (MAP3Ks).TAO2 is a MAP3Klevel kinase that activates the MAP2Ks MEK3 and MEK6 to activate p38 MAPKs.Because p38 MAPKs arekey regulators of expression of inflammatory cytokines,they appear to be involved in human diseases suchas asthma and autoimmunity.As an upstream activator of p38s,TAO2 represents a potential drug target.Here we report the crystal structure of active TAO2 kinase domain in complex with staurosporine,a broad-range protein kinase inhibitor that inhibits TAO2 with an IC_(50) of 3 μM.The structure reveals that staurosporineoccupies the position where the adenosine of ATP binds in TAO2,and the binding of the inhibitor mimicsmany features of ATP binding.Both polar and nonpolar interactions contribute to the enzyme-inhibitorrecognition.Staurosporine induces conformational changes in TAO2 residues that surround the inhibitormolecule,but causes very limited global changes in the kinase.The structure provides atomic details forTAO2-staurosporine interactions,and explains the relatively low potency of staurosporine against TAO2.The structure presented here should aid in the design of inhibitors specific to TAO2 and related kinases.     

Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway

Antibodies that recognise the active phosphorylated forms of mitogen-activated protein kinase (MAPK) kinase 5 (MKK5) and extracellular signal-regulated kinase 5 (ERK5) in untransfected cells have been exploited to show that the epidermal growth factor (EGF)-induced activation of MKK5 and ERK5 occurs subsequent to the activation of ERK1 and ERK2 in HeLa cells. The drugs U0126 and PD184352, which prevent the activation of MKK1 (and hence the activation of ERK1/ERK2), also prevent the activation of MKK5, although higher concentrations are required. Our studies define physiological targets of the MKK5/ERK5 pathway as proteins whose phosphorylation is largely prevented by 10 microM PD184352, but unaffected by 2 microM PD184352. Surprisingly, 2 microM PD184352 prolongs the activation of MKK5 and ERK5 induced by EGF or H(2)O(2), indicating negative control of the MKK5/ERK5 pathway by the classical MAPK cascade. Our results also indicate that ERK5 is not a significant activator of MAPK-activated protein kinase-1/RSK in HeLa cells.     

Expression of human cystatin A by keratinocytes is positively regulated via the Ras/MEKK1/MKK7/JNK signal transduction pathway but negatively regulated via the Ras/Raf-1/MEK1/ERK pathway.

Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form of ras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1, MEK1, ERK1, ERK2, or wild-type MEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1, ERK1, ERK2, or dominant negative forms of MEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (-272 to -278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, or p38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK.     

Downregulation of human endothelial nitric oxide synthase promoter activity by p38 mitogen-activated protein kinase activation.

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. eNOS gene expression may be upregulated by a signaling pathway, including PI-3Kgamma--> Jak2--> MEK1 --> ERK1/2--> PP2A. It remains unclear whether other mitogen-activated protein kinase (MAPK) family members, such as JNK, p38 kinase, and ERK5/BMK1, also modulate eNOS gene expression. Our purpose, therefore, is to shed light on the effect of the p38 MAPK signaling pathway on the regulation of eNOS promoter activity. The results showed that a red fluorescent protein reporter gene vector containing the full length of the human eNOS promoter was first successfully constructed, expressing efficiently in ECV304 cells with the characteristics of real time observation. The wild-types of p38alpha, p38beta, p38gamma, and p38delta signal molecules all markedly downregulated promoter activity, which could be reversed by their negative mutants, including p38alpha (AF), p38beta (AF), p38gamma (AF), and p38delta (AF). Promoter activity was also significantly downregulated by MKK6b (E), an active mutant of an upstream kinase of p38 MAPK. The reduction in promoter activity by p38 MAPK could be blocked by treatment with a p38 MAPK specific inhibitor, SB203580. Moreover, the activation of endogenous p38 MAPK induced by lipopolysaccharide resulted in a prominent reduction in promoter activity. These findings strongly suggest that the activation of the p38 MAPK signaling pathway may be implicated in the downregulation of human eNOS promoter activity.     

Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2

Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.     

Multiple phenotypes resulting from a mutagenesis screen for pharynx muscle mutations in Caenorhabditis elegans

We describe a novel screen to isolate pharyngeal cell morphology mutants in Caenorhabditis elegans using myo-2::GFP to rapidly identify abnormally shaped pharynxes in EMS (Ethyl Methanesulfonate) mutagenized worms. We observed over 83 C. elegans lines with distinctive pharyngeal phenotypes in worms surviving to the L1 larval stage, with phenotypes ranging from short pharynx, unattached pharynx, missing cells, asymmetric morphology, and non-adherent pharynx cells. Thirteen of these mutations have been chromosomally mapped using Single Nucleotide Polymorphisms (SNPs) and deficiency strain complementation. Our studies have focused on genetically mapping and functionally testing two phenotypes, the short pharynx and the loss of muscle cohesion phenotypes. We have also identified new alleles of sma-1, and our screen suggests many genes directing pharynx assembly and structure may be either pharynx specific or less critical in other tissues.     

p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression

Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor beta-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38alpha by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b-->p38alpha blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.     

The Caenorhabditis elegans ortholog of human PHF5a shows a muscle-specific expression domain and is essential for C. elegans morphogenetic development

We have recently described a novel human and murine multigene-family that is highly conserved during evolution and shows a PHD-finger-like domain present in the deduced protein sequences. Here, we describe the cloning and characterization of the Caenorhabditis elegans ortholog of human PHF5a. Transgenic phf-5::yfp-reporter techniques in C. elegans identified temporal C. elegans phf-5 expression being restricted to late C. elegans development. The phf-5::yfp expression starts within the morphogenetic phase of embryonic development and lasts to the stage of adult worms. Spatial phf-5 expression is muscle-specific with an expression in the developing pharynx, in body wall muscular structures, and in the anal muscles. By phf-5 RNAi we further demonstrated that PHF-5 is essential in the morphogenetic phase of C. elegans embryonic development as well as in young larvae. In contrast, phf-5 RNAi does not show an evident phenotype to adult worms. Taken together, this is the first report providing evidence for a tissue and stage-specific expression of a PHF5a ortholog, named phf-5, in C. elegans while our data further suggest an essential role of the encoded PHF-5 protein in morphogenetic development and muscle function.