Kornfeld K et al. (2000) Midwest Worm Meeting "Molecular and Genetic Analysis of lin-1 mutations."

Kelly GR, Kornfeld K

[Midwest Worm Meeting, 2000]

The formation of the C. elegans hermaphrodite vulva is controlled by multiple signaling pathways. The highly conserved RAS pathway promotes the primary vulval cell fate by negatively regulating the ETS transcription factor LIN-1. Bob Horvitz and colleagues isolated 49 alleles of lin-1 in a variety of screens for vulval defects. These alleles include loss-of-function and gain-of-function mutations of different severity. The molecular lesions responsible for some alleles have been identified, but many have yet to be characterized. To identify residues and domains that are important for LIN-1 structure and function, we are using DNA sequencing to determine the molecular lesions of these alleles.

Kornfeld K et al. (1995) International C. elegans Meeting "SUPPRESSORS OF THE let-60 ras MULTIVULVA PHENOTYPE"

Kornfeld K, Horvitz HR, Hom DB

[International C. elegans Meeting, 1995]

An activating mutation in let-60 ras causes P3.p, P4.p and P8.p to adopt vulval fates, resulting in a Multivulva (Muv) 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 Muv phenotype. Forty-three independent mutations were identified. These mutations define 21 complementation groups. Three of these genes are predicted to encode proteins similar to protein kinases that function downstream of Ras in vertebrates and Drosophila. One is the previously identified lin-45 raf gene. Seven suppressor mutations defined a new gene that we named mek-2, because its predicted protein product is most similar to MEK, a protein-serine/threonine and tyrosine kinase. mek-2 mutations can be arranged in an allelic series. A probable null mutation eliminates vulval induction, and the strongest mutations alter codons conserved in most or all protein kinases. One suppressor mutation defined the gene mpk-1/sur-1, a MAP kinase homolog. lin-45, mek-2 and mpk-1 appear to function downstream of let-60 and upstream of lin-1. 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 alleles of a previously unidentified gene that we named sar-2 (suppressor of activated ras). We cloned sar-2 using RFLP mapping and transformation rescue. The predicted SAR-2 protein appears to be a novel protein kinase. We are currently investigating how sar-2 functions during vulval development.

Kornfeld K et al. (1993) International C. elegans Meeting "Suppressors of the let-60 ras multivulva phenotype."

Kornfeld K, Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1993]

Kornfeld K et al. (2000) Midwest Worm Meeting "A Screen for Mutations that Affect Caenorhabditis elegans Aging"

Huang C, Kornfeld K

[Midwest Worm Meeting, 2000]

How aging is controlled is a fundamental biological question that remains largely unanswered. C. elegans promises to be a powerful model system for studying the genetic basis of aging, as it ages rapidly and mutations in genes such as age-1 and daf-2 have been shown to significantly extend the lifespan. We have initiated a screen for mutations that delay the aging process. We characterized the average time for N2 worms to cease egg-laying, display significantly reduced body movement and pharyngeal pumping, and die. Using EMS mutagenesis, we are conducting an F2 clonal screen for mutants with a persistence of the youthful condition for one or more of these processes. We have screened about 1000 (haploid) genomes and identified several mutations that significantly delay one or more of these senescence traits.

Kornfeld K et al. (2000) Midwest Worm Meeting "CDF-1 positively regulates ras-mediated signaling during vulval development"

Kornfeld K, Jakubowski J

[Midwest Worm Meeting, 2000]

A conserved receptor tyrosine kinase-Ras-MAP kinase signal transduction cascade is required for specification of the C. elegans vulva. To identify genes involved in Ras-mediated signaling, we screened for mutations that suppressed the multivulva (Muv) phenotype caused by a constitutively active let-60 ras gene. We isolated 43 mutations that define 21 complementation groups, including the mutation n2527. n2527 reduces the penetrance of the Muv phenotype caused by a let-23(gf) mutation, but not a lin-1(lf) mutation. If these genes act in a linear pathway, the gene affected by n2527 functions downstream of let-23 and let-60 ras and upstream of lin-1. In an otherwise wild-type background, n2527 caused subtle defects in lineages of the vulval precursor cells; this weak vulvaless phenotype is enhanced in a lin-1(gf) mutant. These findings suggest that the gene affected by n2527 functions during normal vulval formation. Mutants containing n2527 in trans to a deficiency displayed a phenotype similar to mutants homozygous for n2527, indicating that n2527 is a loss-of-function mutation. Thus, the gene affected by n2527 appears to be a positive regulator of Ras signaling. Three factor mapping experiments positioned n2527 between mup-2 and unc-6 on chromosome X. To positionally clone the gene affected by n2527, we developed a method of high-resolution mapping relative to single-nucleotide polymorphisms (SNPs) identified by DNA sequencing in the RC301 strain. We found approximately one SNP per 1.4 kb in predicted intergenic regions of RC301. By alternating between identifying polymorphisms and mapping, we positioned the n2527 mutation in a 9.6 kb interval. The molecular lesion was identified by sequencing the interval, and the candidate gene was confirmed by transformation rescue. The n2527 mutation affects a previously uncharacterized gene. The predicted protein has 519 amino acids and is similar to members of the Cation Diffusion Facilitator (CDF) family of proteins. We named this gene cdf-1. CDF-1 is most similar to mammalian ZnT-1 and yeast COT1 which function to reduce the intracellular concentration of Zn2+ and Co2+ ions, respectively. CDF proteins have not been previously shown to be involved in signal transduction. To determine whether CDF-1 has "switch-like" properties characteristic of signaling molecules, we examined the effect of overexpressing cdf-1 in transgenic worms. Overexpression of cdf-1 increased the penetrance of the let-60 ras Muv phenotype, indicating that additional CDF-1 activity can enhance Ras signaling. These findings suggest the possibility that CDF-1 activity is regulated during Ras signaling and that a heavy metal ion may function as a second messenger.

Kornfeld K et al. (2000) Midwest Worm Meeting "Analysis of New Genes Involved in Ras-mediated Signaling During Vulval Induction"

Kornfeld K, Goldstein JL

[Midwest Worm Meeting, 2000]

The development of the C. elegans vulva requires the activity of the Ras signaling pathway. To identify components of this pathway, we screened for mutations that suppress the multivulva phenotype caused by let-60 (n1046gf), a mutation that constitutively activates the ras gene. Forty-three alleles comprising 21 complementation groups were identified. These genes include conserved members of the Ras signaling cascade, such as Raf (lin-45), Ksr (ksr-1), MEK (mek-2), MAP kinase (mpk-1), and an ETS transcription factor (lin-1). We are now analyzing n2528, a mutation that suppresses the let-60 (n1046) muv phenotype. n2528 defines a complementation group that is represented by a single allele. Using high resolution mapping relative to single nucleotide polymorphisms, we have mapped n2528 to a 41 kilobase interval on LG X. To clone the gene affected by n2528, we will refine the map position, determine the sequence of candidate open reading frames in the interval, and attempt transformation rescue. We are also performing genetic analyses to establish how this gene regulates the Ras pathway.

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.

Tan, C.H. et al. (2019) International Worm Meeting "The role of zinc signaling in sperm activation of Caenorhabditis elegans."

Kornfeld, K., Tan, C.H.

[International Worm Meeting, 2019]

Zinc is an essential trace metal. It is the cofactor of many enzymes, and it serves as a structural ion of many transcription factors. Zinc has also been shown to function as a signaling molecule. However, its signaling role has not been well characterized, and the mechanisms remain unclear. In C. elegans, sperm activation is a rapid process that results in the transformation of a round, immotile spermatid into a spermatozoon that crawls with a pseudopod. It is a cell differentiation process with no transcription involved as the nucleus is inaccessible at this stage. Zinc is one of several molecules that can activate C. elegans sperm in vitro. We showed that the zinc transporter ZIPT-7.1 is involved in the sperm activation process. zipt-7.1 is likely to function at a downstream position in the spe-8 sperm activation pathway. Based on these findings, we hypothesize that during sperm activation zinc is released from intracellular vesicles through ZIPT-7.1. This release increases the cytoplasmic zinc concentration, which serves as a signal for further differentiation. To test this hypothesis, we used live imaging and small molecular probes to show that there is a detectable change in the cytoplasmic zinc level during sperm activation. In addition, a zinc ionophore activated sperm in vitro, bypassing the requirement of zipt-7.1 as well as other members of the spe-8 pathway. The results support our model suggesting that zipt-7.1 and the spe-8 pathway activate sperm through zinc signaling. To further understand how sperm are activated by molecules such as zinc and a zinc ionophore, we are currently performing a systematic dose-response analysis of sperm activating efficiency using established sperm activating compounds and different sperm activation mutants.

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.

Jakubowski J et al. (1998) Midwest Worm Meeting "IDENTIFICATION AND CHARACTERIZATION OF GENES THAT FUNCTION DOWNSTREAM OF let-60 ras DURING VULVAL INDUCTION"

Kornfeld K, Jakubowski J

[Midwest Worm Meeting, 1998]

A conserved receptor tyrosine kinase-RAS-MAP kinase signal transduction pathway is required for specification of the vulva. To identify genes involved in vulval induction, we screened for mutations that suppressed the multivulva (Muv) phenotype caused by constitutively active let-60(n1046) ras. Since this Muv phenotype does not require the activity of genes that act upstream of let-60, this screen primarily identified loss-of-function mutations in positively acting downstream genes or gain-of-function mutations in negatively acting downstream genes. We identified 43 mutations including loss-of-function mutations in lin-45 raf, mek-2, mpk-1, sur-2, lin-25, ksr-1 and gain-of-function mutations in lin-1. We have begun analyzing nine mutations that are homozygous viable suppressors of activated Ras and appear to define nine genes we designate sar-5 - sar-13 (suppressor of activated ras). We are mapping these mutations, analyzing their interactions with other genes in the Ras signaling pathway, and performing gene dosage experiments to determine the nature of these alleles. The sar-5(n2527) mutation reduces the penetrance of the lin-15 (lf) Muv phenotype but not the lin-1(lf) Muv phenotype. If these genes act in a linear pathway, sar-5 acts downstream of lin-15 and let-60 ras, and upstream of lin-1 ets. sar-5(n2527) appears to be a gain-of-function mutation, suggesting that sar-5 is a negative regulator of Ras signaling. We speculate that the n2527 mutation results in a constitutively active form of SAR-5 that is not appropriately regulated. To identify loss-of-function mutations in sar-5, we screened for revertants of the sar-5(gf) suppression of let-60(n1046) Muv phenotype. We isolated 33 mutations, including many extragenic suppressors and several candidate intragenic revertants. We have begun a molecular analysis of sar-5 by identifying and mapping sequence polymorphisms between N2 and RC301 in the sar-5 region (near unc-6 X).