Cell Fate Determination and Zinc Metabolism

I. Receptor tyrosine kinase (RTK)-Ras-extracellular-regulated kinase (ERK) MAP kinase pathways are important for animal development and cancer.

Intercellular signaling plays a fundamental role in coordinating different cells during development. Our research is focused on a RTK-Ras-ERK signal transduction pathway that has been highly conserved during evolution and controls many different cell fate choices during the development of vertebrates, Caenorhabditis elegans, and Drosophila.

These pathways play important roles in human diseases. Genes in this signaling pathway, when mutated, are a common cause of human tumors. For example, 10-15% of the most common human tumors contain activating mutations of the ras gene. Several of these signaling proteins were actually discovered as oncogenes, such as receptor tyrosine kinases, Ras and Raf. Thus, an understanding of the function of this signaling pathway is likely to provide a foundation of knowledge that may lead to new diagnostic and therapeutic approaches for cancer.

II. The development of the C. elegans vulva is an important model system for characterizing the RTK-Ras-ERK signaling pathway.

The use of C. elegans to analyze cell fate determination was pioneered by Brenner, Sulston, and Horvitz. These investigators characterized the post-embryonic cell lineage of C. elegans and appreciated that it is essentially invariant from individual to individual. In addition, they identified genes that are necessary for the normal cell lineage, designated lineage-defective (lin). The combination of a fully defined cell lineage and powerful genetic approaches makes C. elegans an important animal model for the analysis of the signal transduction pathways that regulate cell fate determination during development.

As a model system for cell fate determination, Horvitz and colleagues focused on the formation of the vulva, a specialized epithelial structure used for egg-laying and sperm entry. The vulva is well suited for such studies because it is formed during the final larval stages and it is not necessary for adult viability, so that it is possible to recover and propagate mutants with severe defects in vulval morphology. Because vulval formation requires a series of intercellular signaling events, mutations that affect vulval formation frequently affect signaling, and the analysis of vulval formation has led to the discovery and characterization of many signaling proteins. The following cellular events occur during vulval formation (reviewed in Kornfeld, 1997).

In third larval stage hermaphrodites, six ventral epidermal blast cells called P3.p, P4.p, P5.p, P6.p, P7.p and P8.p (Pn.p cells) lie along the anterior-posterior axis (Figure 1A). Each of these Pn.p cells can adopt any of three distinct fates: the primary (1°) vulval cell fate (eight descendants), the secondary (2°) vulval cell fate (seven descendants), or the non-vulval tertiary (3°) cell fate (two descendants). The anchor cell of the somatic gonad signals P6.p to adopt the 1° fate. P6.p signals P5.p and P7.p to adopt the 2° fate by activating lin-12, which is similar to the receptor Notch. P3.p, P4.p and P8.p receive neither signal and adopt the 3° fate (Figure 1B). The 22 descendants of P5.p, P6.p and P7.p differentiate and invaginate to form the vulval structure.

Mutations that reduce the transmission of the anchor cell signal cause all six Pn.p cells to adopt the non-vulval 3° fate, resulting in a vulvaless (Vul) phenotype. By contrast, mutations that result in constitutive activity of the anchor cell signal cause all six Pn.p cells to adopt the 1° or 2° vulval fate, resulting in a multivulva (Muv) phenotype characterized by ectopic patches of vulval tissue. These phenotypes are dramatic, and thus the extent of vulval induction can serve as an easily visualized readout of Ras pathway activity (Figure 1C).

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Figure 1. Vulval development.

The analysis of the anchor cell signal to P6.p has led to many fundamental insights into the function of the RTK-Ras-ERK signaling pathways during development. Some of the notable discoveries include the identification of the anchor cell ligand encoded by lin-3, which is similar to epidermal growth factor, the discovery of the receptor in P6.p encoded by let-23, which is similar to epidermal growth factor receptor, and the discovery of the SH2-SH3-SH2 domain adaptor encoded by sem-5. SEM-5 was the first protein with this domain structure implicated in the Ras signaling pathway (Figure 2).

III. A genetic screen for genes involved in Ras-mediated signaling during vulval formation.

To identify genes that play a role in Ras-mediated signaling, we performed a forward screen for genes that interact with let-60 ras. A gain-of-function (gf) mutation of let-60 ras causes a Muv phenotype, since the fates of P3.p, P4.p and P8.p are transformed to the 1 vulval cell fate. We reasoned that genes that function downstream of let-60 ras would be required for the expression of this Muv phenotype, and such genes could be identified by screening for mutations that affect the Muv phenotype. let-60(gf) mutants that display the Muv phenotype were subjected to random chemical mutagenesis, and 40 mutations that suppress the Muv phenotype were isolated. In principle these could be loss-of-function mutations in genes that promote Ras signaling or gain-of-function mutations in genes that inhibit Ras signaling. Genetic mapping and complementation experiments demonstrated that these 40 mutations identified 20 different genes including the core signaling genes lin-45 raf, mek-2, mpk-1, the modulatory signaling genes ksr-1, cdf-1, sur-8 and cgr-1, and the transcriptional response genes lin-1, sur-2 and lin-25.

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Figure 2. Conserved RTK-Ras-ERK
signaling pathway.

IV. Studies of the core signaling proteins RAF, MEK, and ERK.

We identified six lin-45 raf alleles in the screen for suppressors of the let-60 ras Muv phenotype. Genetic studies were used to classify these alleles as weak, intermediate, or strong, and molecular studies revealed missense mutations in important functional domains. These studies were led by Virginia Hsu, an undergraduate student (Hsu et al., 2002). To investigate the biochemical defects caused by these missense mutations, we engineered these changes into the homologous residues of vertebrate Raf-1 and expressed the mutant Raf proteins in vertebrate cells. These studies, which were conducted in collaboration with the laboratory of John Hancock, identified biochemical functions of Raf that are important for Raf activity in animals (Harding et al., 2003).

We identified seven alleles of a previously uncharacterized gene that encoded the C. elegans homolog of the MAP kinase kinase MEK, and thus we named the gene mek-2. These studies provided important in vivo genetic evidence that MEK functions together with Raf and ERK (Kornfeld et al., 1995).

We identified one allele of a previously uncharacterized gene that encoded a protein homologous to vertebrate ERK MAP kinase. Thus, we named the gene mpk-1. These studies were a collaboration with the laboratory of Stuart Kim and provided in vivo genetic evidence that ERK plays a critical role in the Ras pathway (Lackner et al., 1994). We began a biochemical investigation of ERK interactions with substrate proteins as part of the analysis of the LIN-1 ETS transcription factor. We identified six gain-of-function mutations of lin-1, and we noted that each mutation affected an FQFP motif (Jacobs et al., 1998). We hypothesized that the FQFP motif functioned as a docking site that mediated interactions of ERK with specific substrates. Biochemical studies showed that the FQFP motif was necessary for high affinity interactions, and FQFP was sufficient to increase the affinity of non-specific substrates for ERK. A second docking site called the D domain and the FQFP motif function independently and additively to mediate a high-affinity interaction. The analysis of ERK docking sites provides information that can be used to predict ERK substrates based on the amino acid sequence of the protein. For example, we predicted an interaction between KSR and ERK based on the presence of an FXFP motif in KSR. These studies were led by David Jacobs, a research associate (Jacobs et al., 1999). To define the recognition determinants in the FQFP motif, we performed extensive mutagenesis studies that were led by Douglas Fantz, a postdoctoral fellow (Fantz et al., 2001). Based on these studies we concluded that the docking sites form a modular system that regulates the affinity of the enzyme-substrate interaction and directs phosphorylation of specific S/TP residues.

V. The LIN-1 ETS transcription factor is a critical target of Ras-mediated signaling and an important model system for vertebrate Elk-1.

Genetic studies demonstrate that lin-1 functions at the end of the signaling pathway and plays a critical role in controlling vulval cell fates. lin-1 encodes a protein with an ETS domain that is predicted to bind DNA and regulate transcription. We demonstrated that the LIN-1 ETS domain has sequence-specific DNA binding activity. Mutations that disrupt DNA binding cause a loss of lin-1 activity, indicating that DNA binding is necessary for lin-1 function. These studies were led by Ginger Miley, a graduate student (Miley et al., 2004). We identified a target gene that directly binds LIN-1 and responds to LIN-1 activity in vivo in collaboration with the laboratory of David Eisenmann (Wagmaister et al., 2006.). We discovered that LIN-1 is modified by phosphorylation and sumoylation, and we have focused on understanding how these modifications control LIN-1 activity. We demonstrated that LIN-1 has two docking sites for ERK, the D domain and the FQFP motif, and that LIN-1 is a high affinity substrate for ERK. Mutations in the FQFP docking site that abrogate the interaction with ERK result in constitutively active LIN-1 protein that is unresponsive to Ras signaling and causes a Vul phenotype. Thus, the interaction with ERK is necessary to reverse the inhibition of the 1 vulval cell fate caused by LIN-1 (Jacobs et al., 1998, 1999).

To identify proteins that associate with LIN-1 and contribute to the regulation of vulval cell fates, we conducted a yeast two-hybrid screen. We discovered that LIN-1 interacts with Ubc9, an E2 SUMO conjugating enzyme. Small ubiquitin-related modifier (SUMO) is covalently attached to a lysine residue in the protein substrate by a reversible process that is analogous to ubiquitinylation. LIN-1 contains two consensus sites for SUMO attachment, and we demonstrated that LIN-1 is sumoylated using biochemical methods. Cell based assays were used to demonstrate that sumoylation of LIN-1 promotes transcriptional repression. These studies were led by Elizabeth Leight, a postdoctoral fellow (Leight et al., 2005). We have identified four proteins that interact with LIN-1 and appear to mediate transcriptional repression: MEP-1, RAD-26, MAS-1 and EGL-27. As shown in Figure 3, we propose that LIN-1 is converted from a sumoylated transcriptional repressor to a phosphorylated transcriptional activator to control the fate of the vulval precursor cell.
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Figure 3. A model of cell fate regulation by LIN-1.

VI. Identification of the pathway modulators ksr-1, cgr-1 and cdf-1.

We identified six alleles of a previously uncharacterized gene and showed it encoded a novel protein with a predicted protein kinase domain, and thus we named the gene kinase suppressor of ras (ksr-1). An extensive genetic analysis indicated that ksr-1 positively regulates Ras-mediated signaling (Kornfeld et al., 1995). To characterize the biochemical function of C. elegans and vertebrate KSR, we used purified proteins and studies of cultured cells. We demonstrated that KSR interacts with 14-3-3, binds Raf and translocates from the cytoplasm to the membrane in response to activation of the Ras signaling pathway. These studies were a collaboration with the laboratory of Anthony Muslin (Xing et al., 1997). We noted that KSR contains FXFP docking sites for ERK. We demonstrated that these docking sites mediate a high-affinity interaction with ERK and that KSR can be phosphorylated by ERK (Jacobs et al., 1999; Fantz et al., 2001). Together, these genetic and biochemical studies suggest the model that KSR is necessary for signal propagation because it functions as a scaffold that facilitates the interaction of Raf, MEK and ERK.

We identified one allele of a previously uncharacterized gene that encodes a protein with a CRAL/TRIO domain that is likely to bind a small, hydrophobic ligand and a GOLD domain that may mediate protein-protein interactions. Thus, we named the gene CRAL/TRIO and GOLD domain suppressor of Ras (cgr-1). cgr-1 positively regulated Ras signaling, and these studies were led by Jessica Goldstein, a graduate student (Goldstein et al., 2006). A bioinformatics analysis revealed that there is a conserved family of CRAL/TRIO and GOLD domain containing proteins that includes members from vertebrates, and we have now demonstrated that vertebrate proteins can also modulate Ras signaling. The analysis of cgr-1 identifies a novel in vivo function for a member of this family and a new regulator of Ras-mediated signaling.

We identified one allele of a previously uncharacterized gene; to clone the gene, we developed methods for generating a local, high density, single-nucleotide polymorphism map and positioned the mutation to a small interval. This project was led by Janelle Jakubowski Bruinsma, a graduate student (Jakubowski and Kornfeld, 1999). The gene encodes a novel protein that is homologous to cation diffusion facilitator proteins. Thus, we named the gene cdf-1. The cdf-1 gene functions downstream of Ras and upstream of ERK to positively mediate Ras signaling. CDF proteins promote zinc efflux and reduce the concentration of cytosolic zinc. This finding suggested the novel hypothesis that cytosolic zinc negatively regulates Ras-mediated signaling. In support of this hypothesis, we showed that feeding zinc to worms or injection of zinc into Xenopus cells inhibits Ras signaling. These findings suggest that zinc negatively regulates a conserved element of the signaling pathway and that zinc regulation is important for maintaining the inactive state of the Ras pathway (Bruinsma et al., 2002).

VII. C. elegans as a model for studying zinc metabolism

Zinc metabolism and homeostasis are fundamental biological processes, since zinc is essential for the function of many proteins and zinc modulates signal transduction pathways. Zinc is an essential nutrient that profoundly affects human health, as zinc deficiency and excess both result in a broad spectrum of pathologies. However, mechanisms that mediate zinc metabolism are only beginning to be defined. Having discovered a role for zinc in Ras-mediated signaling, we initiated a research program to understand how a network of zinc transporters and binding proteins act in a coordinated fashion to regulate zinc metabolism in multicellular animals. We are addressing this by analyzing zinc importers, zinc exporters and new genes involved in zinc metabolism using the genetically tractable model organism C. elegans. Understanding how a network of proteins controls zinc metabolism in a multicellular animal is an important objective of medical research, since the information may lead to new therapeutic approaches for diseases caused by abnormal zinc metabolism.

The regulation of Zn² ⁺ in a multicellular organism can be considered at two different levels of organization (Figure 4). Individual cells in a multicellular organism must have mechanisms to control Zn² ⁺-uptake, Zn² ⁺-secretion, and the concentration of Zn² ⁺ in the cytoplasm and organelles. A second level of organization is the entire animal. A multicellular animal must take up dietary Zn² ⁺, distribute that Zn² ⁺ throughout the body, and excrete excess Zn² ⁺. These processes represent unique challenges that are not confronted by single-cell organisms. The understanding of this level of organization remains primitive, and advances will require a multicellular model organism that is experimentally tractable.

We have established the powerful C. elegans model system for studies of zinc metabolism by developing culture conditions that permit manipulation of dietary zinc, establishing assays of zinc metabolism, and identifying new genes that affect zinc metabolism. Dietary zinc can be manipulated precisely by culturing in defined liquid medium, and growth, development and reproduction are sensitive to zinc deficiency and zinc excess. The zinc content of worms can be quantified using radioactive zinc or mass spectrometry, and zinc content can be dramatically affected by dietary zinc or the activity of cdf genes. Current studies of zinc metabolism are focused on three areas: identification of genes that influence resistance to high dietary zinc, transcriptional control by dietary zinc, and the function of the CDF family of zinc transporters.

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Figure 4. A model of zinc transport
in an animal.

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Figure 5. Identification of 19 mutations that
cause resistance to high dietary zinc.

To identify genes involved in zinc metabolism and Ras signaling, we conducted a forward genetic screen for chemically induced mutations that cause C. elegans to be resistant to high levels of dietary zinc. Nineteen mutations were identified that confer significant resistance to supplemental dietary zinc (Figure 5). Mapping experiments indicate that the nineteen mutations identify at least five genes involved in zinc metabolism. This project was led by Janelle Bruinsma, a postdoctoral fellow (Bruinsma et al., 2008). We molecularly identified one of these genes, and it encodes a protein that appears to be involved in histidine metabolism (Figure 6). We are planning to molecularly identify the additional genes using a combination of mapping and DNA sequencing approaches.
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Figure 6. A gene involved in histidine
metabolism causes resistance
to high dietary zinc.
To determine how zinc regulates gene expression, we analyzed transcriptional responses to dietary zinc. We established conditions to completely control dietary zinc using a defined liquid medium called CeMM. We cultured worms in a wide range of dietary zinc and analyzed transcription by quantitative PCR. By testing candidate genes, we identified several genes that are transcriptionally regulated by zinc including the C. elegans metalothionein genes (mtl-1 and mtl-2) and two members of the cation diffusion facilitator family (cdf-2 and ttm-1a). mtl-1 and mtl-2 show dramatic increases in RNA abundance in response to dietary zinc, varying from 100 to 1000 fold (Figure 7, A-B). To identify the cis regulatory elements responsible for this transcriptional control, we established an assay in transgenic animals. We generated plasmids containing the mtl genes fused to a reporter gene called green fluorescent protein, introduced these plasmids into worms to create transgenic animals, and monitored RNA and protein induction by dietary zinc. We have identified promoter fragments that are sufficient to cause gene induction, and we are currently performing mutagenesis to determine the elements that are responsible for transcriptional control (Figure 7, C-F). We plan to define the elements that are necessary and sufficient to mediate induction of transcription. To identify factors that can act in trans to affect transcription, we plan to conduct a genetic screen for mutants with abnormal transcriptional responses using the green fluorescent protein as a reporter. In addition to analyzing candidate genes, we have used microarray technology to discover genes that are regulated by zinc and likely play a role in zinc metabolism.
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Figure 7. Dietary zinc regulates
gene expression.

To identify additional CDF family members that affect zinc metabolism, we used a bioinformatics analysis to identify all the C. elegans CDF proteins. There are 14 predicted CDF family members in C. elegans. We plan to analyze the function of these proteins, and we have begun obtaining loss-of-function mutations in these genes. We characterized the gene T18D3.3, which we named cdf-2. This project was led by Diana Davis, a graduate student (Davis et al., 2009). CDF-2 protein is localized to vesicles in the intestinal cells, indicating it plays a role in zinc storage. Using the chemically defined media, we analyzed double and triple mutant combination of cdf-2, cdf-1 and sur-7. We established that these genes interact, and cdf-1 and cdf-2 functionantagonistically to influence zinc content (Figure 8).



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Figure 8. A model of CDF-1 and CDF-2 activity.

Bruinsma, J. J., T. Jirakulaporn, A. J. Muslin and K. Kornfeld. 2002. Zinc ions and cation diffusion facilitator proteins regulate Ras-mediated signaling. Developmental Cell 2: 567-578.

Bruinsma, J.J., Schneider D., Davis, D., and K. Kornfeld. 2008. Identification of mutations in Caenorhabditis elegans that cause resistance to high levels of dietary zinc and analysis using a genome-wide map of single-nucleotide polymorphisms scored by pyrosequencing. Genetics, 179: 811-828.

Davis, D.E., Roh, H.C., Deshmukh, K., Bruinsma, J.J., Schneider, D.L., Guthrie, Robertson, J.D., and K. Kornfeld. 2009. The Cation Diffusion Facilitator Gene cdf-2 Mediates Zinc Metabolism in Caenorhabditis elegans. Genetics, 182: 1015–1033.

Fantz, D.A., D. Jacobs, D. Glossip and K. Kornfeld. 2001. Docking sites on substrate proteins direct extracellular-signal regulated kinase to phosphorylate specific residues. J. Biol. Chem. 276: 27256-27265.

Goldstein, J., D. Glossip, S. Nayak and K. Kornfeld. 2006. The CRAL/TRIO and GOLD domain protein CGR-1 promotes induction of vulval cell fates in Caenorhabditis elegans and interacts genetically with the Ras signaling pathway. Genetics, 172: 929-942.

Harding, A., V. Hsu, K. Kornfeld and J.F. Hancock. 2003. Identification of residues and domains of Raf important for function in vivo and in vitro. J. Biol. Chem. 278: 45519-45527.

Hsu, V., Zobel, C., Lambie, E., Schedl. T. and K. Kornfeld. 2002. Caenorhabditis elegans lin-45 raf is essential for larval viability, fertility and the induction of vulval cell fates. Genetics 160: 481-492.

Jacobs, D., G.J. Beitel, S.G Clark. H.R. Horvitz and K. Kornfeld. 1998. Gain-of-function mutations in the C. elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase. Genetics 149: 1809-1822.

Jacobs, D., D. Glossip, H. Xing, A.J. Muslin and K. Kornfeld. 1999. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes & Development 13:163-175.

Jakubowski, J., and K. Kornfeld. 1999. A local, high-density, single-nucleotide polymorphism map used to clone Caenorhabditis elegans cdf-1. Genetics 153: 743-752.

Kornfeld, K., K.-L. Guan and H.R. Horvitz. 1995. The Caenorhabditis elegans gene mek-2 is required for vulval induction and encodes a protein similar to the protein kinase MEK. Genes & Development 9: 756-768.

Kornfeld, K., D.B. Hom and H.R. Horvitz. 1995. The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans. Cell 83: 903-913.

Kornfeld, K. 1997. Vulval development in Caenorhabditis elegans. Trends in Genetics 13: 55-61.

Lackner, M.R., K. Kornfeld, L.M. Miller, H.R. Horvitz and S.K. Kim. 1994. A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans. Genes & Development 8: 160-173.

Leight, E., D. Glossip, and K. Kornfeld. 2005. Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates. Development 132: 1047-1056.

Miley, G., D. Fantz, D. Glossip, X. Lu, R. Saito,, R. Palmer, T. Inoue, S. van den Heuvel, P. Sternberg, and K. Kornfeld. 2004. Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates. Genetics 167: 1697-1709.

Wagmaister, J. A., G.R. Miley, C.A. Morris, J.E. Gleason, L.M. Miller, K. Kornfeld and D. M. Eisenmann. 2006. Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31, and LIN-39. Developmental Biology 297: 550-565.

Xing, H., K. Kornfeld and A.J. Muslin. 1997. The protein kinase KSR interacts with 14-3-3 protein and Raf. Current Biology 7: 294-300.







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