Kerry Kornfeld

The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans

By screening for mutations that suppress the vulval defects caused by a constitutively active let-60 ras gene, we identified six loss-of-function alleles of ksr-1, a novel C. elegans gene. 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 recently identified Drosophila protein involved in Ras signaling. We propose that the function of ksr-1 is evolutionarily conserved.

The protein kinase KSR interacts with 14-3-3 protein and Raf

BACKGROUND KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster. The function of vertebrate KSR is not well characterized biochemically or biologically. RESULTS We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3. Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction. CONCLUSIONS Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.

Vulval development in Caenorhabditis elegans

Ever since the cell lineage of the nematode Caenorhabditis elegans was shown to be nearly invariant, investigators have tried to understand the mechanisms that control these precise patterns of cell divisions and cell fates. Important insights have come from analyzing the cells that form the hermaphrodite vulva, a specialized hypodermal passageway used for egg laying and sperm entry. Early experiments showed that the invariant pattern of vulval cell fates requires highly reproducible intercellular signals. This review describes recent experiments that have begun to characterize molecules that mediate these signals and explore the relationships between different signaling pathways. Many of these molecules and signaling pathways have been conserved during evolution suggesting mechanisms used to establish patterns of cell fates during vulval development have also been conserved.

Genetic and pharmacological factors that influence reproductive aging in nematodes.

Age-related degenerative changes in the reproductive system are an important aspect of aging, because reproductive success is the major determinant of evolutionary fitness. Caenorhabditis elegans is a prominent organism for studies of somatic aging, since many factors that extend adult lifespan have been identified. However, mechanisms that control reproductive aging in nematodes or other animals are not well characterized. To use C. elegans to measure reproductive aging, we analyzed mated hermaphrodites that do not become sperm depleted and monitored the duration and level of progeny production. Mated hermaphrodites display a decline of progeny production that culminates in reproductive cessation before the end of the lifespan, demonstrating that hermaphrodites undergo reproductive aging. To identify factors that influence reproductive aging, we analyzed genetic, environmental, and pharmacological factors that extend lifespan. Dietary restriction and reduced insulin/insulin-like growth factor signaling delayed reproductive aging, indicating that nutritional status and a signaling pathway that responds to environmental stress influence reproductive aging. Cold temperature delayed reproductive aging. The anticonvulsant medicine ethosuximide, which affects neural activity, delayed reproductive aging, indicating that neural activity can influence reproductive aging. Some of these factors decrease early progeny production, but there is no consistent relationship between early progeny production and reproductive aging in strains with an extended lifespan. To directly examine the effects of early progeny production on reproductive aging, we used sperm availability to modulate the level of early reproduction. Early progeny production neither accelerated nor delayed reproductive aging, indicating that reproductive aging is not controlled by use-dependent mechanisms. The implications of these findings for evolutionary theories of aging are discussed.

Zinc Ions and Cation Diffusion Facilitator Proteins Regulate Ras-Mediated Signaling

C. elegans cdf-1 was identified in a genetic screen for regulators of Ras-mediated signaling. CDF-1 is a cation diffusion facilitator protein that is structurally and functionally similar to vertebrate ZnT-1. These proteins have an evolutionarily conserved function as positive regulators of the Ras pathway, and the Ras pathway has an evolutionarily conserved ability to respond to CDF proteins. CDF proteins regulate Ras-mediated signaling by promoting Zn(2+) efflux and reducing the concentration of cytosolic Zn(2+), and cytosolic Zn(2+) negatively regulates Ras-mediated signaling. Physiological concentrations of Zn(2+) cause a significant inhibition of Ras-mediated signaling. These findings suggest that Zn(2+) negatively regulates a conserved element of the signaling pathway and that Zn(2+) regulation is important for maintaining the inactive state of the Ras pathway.

Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans.

The identification and analysis of compounds that delay aging and extend lifespan is an important aspect of gerontology research; these studies can test theories of aging, lead to the discovery of endogenous systems that influence aging, and establish the foundation for treatments that might delay normal human aging. Here we review studies using the nematode Caenorhabditis elegans to identify and characterize compounds that delay aging and extend lifespan. These studies are considered in four groups: (1) Studies that address the free-radical theory of aging by analyzing candidate compounds with antioxidant activities including vitamin E, tocotrienols, coenzyme Q, and Eukarion-8/134. (2) Studies that analyze plant extracts (blueberry and Ginko biloba) that contain a mixture of compounds. (3) Studies of resveratrol, which was identified in a screen for compounds that affect the activity of the Sir2 protein that influences lifespan. (4) Studies based on screening compound libraries using C. elegans aging as a bioassay, which led to the identification of the anticonvulsant medicines ethosuximide and trimethadione. There has been exciting progress in the analysis of compounds that influence C. elegans aging, and important challenges and opportunities remain in determining the mechanisms of action of these compounds and the relevance of these observations to aging of other animals.

The measurement and analysis of age-related changes in Caenorhabditis elegans.

Aging is characterized by progressive degenerative changes in tissue organization and function that increase the probability of mortality. Major goals of aging research include elucidating the series of events that cause degenerative changes and analyzing environmental and genetic factors that modulate these changes. The basis for mechanistic studies of aging are accurate and precise descriptions of age-related changes, since these descriptions define the aging phenotype. Here we review studies that describe age-related changes in C. elegans including measurements of integrated functions such as behavior, microscopic analyses of tissue organization, and biochemical studies of macromolecules. Genetic and environmental factors that influence these changes are described, and studies that analyze the relationships between different age-related changes are discussed. Together these studies provide fundamental insights into aging in C. elegans that may be relevant to aging in other animals.

Valproic acid extends Caenorhabditis elegans lifespan

Aging is an important biological phenomenon and a major contributor to human disease and disability, but no drugs have been demonstrated to delay human aging. Caenorhabditis elegans is a valuable model for studies of animal aging, and the analysis of drugs that extend the lifespan of this animal can elucidate mechanisms of aging and might lead to treatments for age‐related disease. By testing drugs that are Food and Drug Administration approved for human use, we discovered that the mood stabilizer and anticonvulsant valproic acid (VA) extended C. elegans lifespan. VA also delayed age‐related declines of body movement, indicating that VA delays aging. Valproic acid is a small carboxylic acid that is the most frequently prescribed anticonvulsant drug in humans. A structure–activity analysis demonstrated that the related compound valpromide also extends lifespan. Valproic acid treatment may modulate the insulin/IGF‐1 growth factor signaling pathway, because VA promoted dauer larvae formation and DAF‐16 nuclear localization. To investigate the mechanism of action of VA in delaying aging, we analyzed the effects of combining VA with other compounds that extend the lifespan of C. elegans. Combined treatment of animals with VA and the heterocyclic anticonvulsant trimethadione caused a lifespan extension that was significantly greater than treatment with either of these drugs alone. These data suggest that the mechanism of action of VA is distinct from that of trimethadione, and demonstrate that lifespan‐extending drugs can be combined to produce additive effects.

Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates

The LIN-1 ETS transcription factor inhibits vulval cell fates during Caenorhabditis elegans development. We demonstrate that LIN-1 interacts with UBC-9, a small ubiquitin-related modifier (SUMO) conjugating enzyme. This interaction is mediated by two consensus sumoylation motifs in LIN-1. Biochemical studies showed that LIN-1 is covalently modified by SUMO-1. ubc-9 and smo-1, the gene encoding SUMO-1, inhibit vulval cell fates and function at the level of lin-1, indicating that sumoylation promotes LIN-1 inhibition of vulval cell fates. Sumoylation of LIN-1 promoted transcriptional repression and mediated an interaction with MEP-1, a protein previously shown to associate with the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies showed that mep-1 inhibits vulval cell fates and functions at the level of lin-1. We propose that sumoylation of LIN-1 mediates an interaction with MEP-1 that contributes to transcriptional repression of genes that promote vulval cell fates. These studies identify a molecular mechanism for SUMO-mediated transcriptional repression.

Identification of Mutations in Caenorhabditis Elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

Zinc plays many critical roles in biological systems: zinc bound to proteins has structural and catalytic functions, and zinc is proposed to act as a signaling molecule. Because zinc deficiency and excess result in toxicity, animals have evolved sophisticated mechanisms for zinc metabolism and homeostasis. However, these mechanisms remain poorly defined. To identify genes involved in zinc metabolism, we conducted a forward genetic screen for chemically induced mutations that cause Caenorhabditis elegans to be resistant to high levels of dietary zinc. Nineteen mutations that confer significant resistance to supplemental dietary zinc were identified. To determine the map positions of these mutations, we developed a genomewide map of single nucleotide polymorphisms (SNPs) that can be scored by the high-throughput method of DNA pyrosequencing. This map was used to determine the approximate chromosomal position of each mutation, and the accuracy of this approach was verified by conducting three-factor mapping experiments with mutations that cause visible phenotypes. This is a generally applicable mapping approach that can be used to position a wide variety of C. elegans mutations. The mapping experiments demonstrate that the 19 mutations identify at least three genes that, when mutated, confer resistance to toxicity caused by supplemental dietary zinc. These genes are likely to be involved in zinc metabolism, and the analysis of these genes will provide insights into mechanisms of excess zinc toxicity.