Jacobs D et al. (1996) Midwest Worm Meeting "Suppressors of the let-60 ras multivulva phenotype"

Kornfeld K, Syder D, Jacobs D

[Midwest Worm Meeting, 1996]

An activating mutation in let-60 ras causes P3.p, P4.p and P8.p to adopt vulval fates, resulting in a multivulva phenotype. To identify additional genes involved in vulval development, particularly genes that act downstream of let-60 ras, we screened for mutations that suppress this phenotype. Forty-three independent mutations were identified that define 21 complementation groups. Nine of these genes have been cloned and characterized genetically by us or others; twelve genes have not, as yet, been characterized in detail. Three of these genes, lin-45, mek-2 and mpk-1/sur-1, are predicted to encode proteins similar to protein kinases that function downstream of Ras in vertebrates and Drosophila. Biochemical analyses of similar vertebrate proteins suggests that LET-60 may bind LIN-45, LIN-45 may then phosphorylate MEK-2, and MEK-2 may then phosphorylate MPK-1 We identified six loss-of-function alleles of ksr-1 (kinase suppressor of ras). Our genetic analysis showed ksr-1 positively mediates Ras signaling and functions downstream of or in parallel to let-60. In the absence of ksr-1 function, normal Ras signaling is impaired only slightly, suggesting ksr-1 may act to modulate or in a branch that diverges from the main signaling pathway. The predicted KSR-1 protein has a protein kinase domain and is most similar to a newly identified Drosophila protein involved in Ras signaling. Thus, the function of ksr-1 is likely to be evolutionarily conserved. We are currently analyzing the distribution of ksr-1 gene products during development. lin-1 is negatively regulated by Ras signaling and acts downstream of mpk-1. lin-1 is predicted to encode a protein that contains an ETS domain suggesting LIN-1 may be a transcription factor. We are investigating how lin-1 is regulated by upstream signaling events.

Jacobs D et al. (1998) Midwest Worm Meeting "LIN-1 ETS IS PHOSPHORYLATED BY ERK MAP KINASE"

Jacobs D, Kornfeld K

[Midwest Worm Meeting, 1998]

LIN-1 encodes a 441 amino acid protein that is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain. lin-1(lf) mutations cause a multivulva phenotype, and lin-1(gf) mutations cause a vulvaless phenotype, suggesting lin-1 is a switch molecule and lin-1 activity prevents Pn.p cells from adopting the primary vulval fate. Genes in the Ras signaling pathway promote the primary fate, and epistasis analyses indicate lin-1 functions downstream of these genes, including mpk-1 Erk MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. To investigate whether LIN-1 is directly regulated by MAP kinase, we produced LIN-1 protein in E. coli and assayed partially purified protein for phosphorylation by murine Erk2 MAP kinase in vitro. Full-length LIN-1 protein was an excellent substrate with a Km of 0.2 uM, significantly lower that the 3.3 uM Km of myelin basic protein, a protein frequently used to assay Erk activity. The C-terminal region of LIN-1 (residues 281-441) was necessary for phosphorylation and sufficient to function as a good Erk2 substrate. The lin-1(gf) mutations all affect the C-terminal region of LIN-1, suggesting this region is required for negative regulation of LIN-1. These observation indicate that phosphorylation of the C-terminal region of LIN-1 by Erk negatively regulates LIN-1 activity. We produced and assayed two mutant LIN-1 proteins that contained the alterations caused by two different lin-1(gf) mutations. These proteins had Km values that were significantly higher the Km of wild-type LIN-1, suggesting the lin-1(gf) mutations function by diminishing LIN-1 phosphorylation by Erk. The lin-1(gf) mutations may affect a recognition motif for Erk MAP kinase.

Jacobs D et al. (1997) International C. elegans Meeting "GAIN-OF-FUNCTION MUTATIONS SUGGEST LIN-1 IS NEGATIVELY REGULATED BY MAP KINASE PHOSPHORYLATION"

Horvitz HR, Jacobs D, Kornfeld K, Beitel GJ

[International C. elegans Meeting, 1997]

lin-1(lf) mutations cause a highly expressive multivulva phenotype suggesting lin-1 is an important negative regulator of the 1! vulval cell fate. Genes in the ras signaling pathway promote the 1! fate, and epistasis analyses suggest lin-1 functions downstream of these genes, including mpk-1 MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. The predicted LIN-1 protein contains an N-terminal ets domain, suggesting LIN-1 may be a DNA-binding transcription factor. We identified four different lin-1 mutations that cause partially penetrant larval lethality characterized by a rod-like morphology and vulval defects typical of a lack of vulval induction. These same phenotypes are caused by loss-of-function mutations in genes in the ras signaling pathway but are the opposite of the phenotypes caused by a loss-of-function mutation in lin-1. Dosage studies suggest that these four lin-1 alleles are gain-of-function mutations. We determined the base changes present in these alleles, and each is predicted to affect the C-terminal region of LIN-1. This region appears to be highly divergent from but nonetheless similar to regions of vertebrate ets domain-containing proteins that are phosphorylated and thereby regulated by MAP kinase. These observations suggest that the C-terminus of LIN-1 is phosphorylated by MAP kinase, that this phosphorylation negatively regulates the activity of LIN-1, and that the mutant LIN-1 proteins have lost the ability to be phosphorylated and thereby negatively regulated but retain the ability to inhibit the 1! vulval cell fate. We are currently testing these biochemical hypotheses.

Jacobs D et al. (1999) Genes Dev "Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase."

Jacobs D, Xing H, Kornfeld K, Muslin A, Glossip D

[Genes Dev, 1999]

MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, forma a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities to ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predicts new EFK substrates and design peptide inhibitors of EFK that functioned in vitro and in vivo.

Jacobs D et al. (1998) Genetics "Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase."

Jacobs D, Beitel GJ, Kornfeld K, Clark SG, Horvitz HR

[Genetics, 1998]

Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. We identified and characterized six gain-of-function mutations that define a new class of lin-1 allele. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting this region is required for negative regulation. The C terminus of LIN-1 was a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, our results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, that is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases.

Kornfeld K et al. (2000) Midwest Worm Meeting "ERK MAP Kinase Recognition of Substrates is Mediated by a Modular System of Docking Sites that Directs Phosphorylation at Specific Residues"

Fantz DA, Kornfeld K, Jacobs D

[Midwest Worm Meeting, 2000]

MAP kinases are signaling molecules that phosphorylate S/TP motifs in a wide variety of substrate proteins. The mechanisms that enable MAP kinases to identify substrate proteins and phosphorylate functionally important residues are not well understood. We are investigating how MAP kinases interact with substrate proteins by focusing on the C. elegans proteins, LIN-1 and KSR-1, and the human ETS transcription factor, Elk-1. LIN-1 and KSR-1 are part of the Ras signaling pathway required for vulval formation in C. elegans. Genetic studies of lin-1 indicate that it is negatively regulated by the Ras pathway and functions downstream of mpk-1 ERK MAP kinase. KSR-1 is a protein kinase that positively mediates Ras signaling. We have shown that LIN-1, KSR-1, and Elk-1 can function as high-affinity substrates for ERK MAP kinase in vitro. All three of these proteins possess an ERK MAP kinase docking site defined by an FXFP motif. The ETS transcription factors LIN-1 and Elk-1 also possess a second type of docking site called the D-box which appears to mediate binding to ERK and JNK MAP kinase. FXFP and the D-box can function independently or in combination as docking sites to form a modular recognition system for MAP kinases. In vitro assays have been used to define the primary sequence that is necessary for FXFP function and to determine the protein context required for FXFP and D-box function. Peptide inhibition assays have demonstrated that the FXFP and D-box sites bind different pockets on the ERK MAP kinase enzyme and possess distinct spatial requirements between the docking site and potential phosphorylation sites. Further in vitro experiments have been performed to characterize a third docking site found in ribosomal S6 kinase that has recently been described to mediate interactions between ERK and p38 MAP kinases and ribosomal S6 kinase. We have also found evidence suggesting that these distinct docking sites mediate preferential phosphorylation of specific S/TP motifs in a substrate. It is known that phosphorylation of Elk-1 at Serine-383 is required for its transcriptional activation. We have shown using phospho-specific antibodies that an FXFP docking site in its wild-type context is necessary for highly efficient phosphorylation at Serine-383 of Elk-1. This suggests that MAP kinases may utilize the modular nature of FXFP and the D box to differentially regulate substrates by directing phosphorylation to functionally distinct sites. We have also demonstrated the relevance of FXFP and D box docking sites as in vivo regulators of ERK MAP kinase/substrate interactions in vertebrate cells by analyzing the transactivation potential of wild-type Elk-1 and variants containing mutations in FXFP and/or the D box. The combination of in vitro enzyme assays and in vivo functional assays have provided a set of guidelines for the prediction of potential in vivo substrates of ERK MAP kinases by defining the sequence and context necessary for the function of MAP kinase docking sites.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.