MAP Kinase Regulation Of Substrate Proteins
Kerry Kornfeld, Associate Professor
Developmental Biologywashington University
Grant 5R01CA084271-08 from National Cancer Institute, IRG: CSD
Abstract: The Ras - ERK MAP kinase signaling pathway is critical for establishing cell fates during development. Importantly, mutations that activate the Ras pathway are a prevalent cause of human tumors. A major gap in our current understanding of this pathway is how activation of ERK alters the balance in gene expression to promote cell proliferation. Our long term goal is to understand how the Ras signaling pathway switches the activity of transcription factors so that they promote cell proliferation. We will address this by analyzing transcription factors that respond to Ras-mediated signaling in C. elegans and vertebrates. Understanding how the Ras pathway controls gene expression in development and disease in an important objective of medical research, since the information may lead to new and effective therapies for tumors caused by activated Ras signaling. Our preliminary results have defined exciting new mechanisms for the regulation of the LIN-1 transcription factor by Ras-mediated signaling and support two innovative hypotheses. (1) The LIN-1 transcription factor is post translationally modified by SUMO. Sumoylated LIN-1 represses transcription by interacting with MEP-1, DAS-1 and MAS-1, proteins involved in chromatin remodeling. (2) Activated ERK switches LIN-1 from a sumoylated, transcriptional represser that inhibits cell proliferation to a phosphorylated, transcriptional activator that induces cell proliferation. To test these hypotheses, we propose three specific aims. Aims 1 and 2 We will use biochemical techniques to define domains and specific residues of MEP-1, MAS-1 and DAS-1 that interact with LIN-1. We will characterize the relevance and specific functions of these interactions in intact animals. These studies will define the mechanisms of SUMO-mediated transcriptional repression and the significance of one DNA binding transcription factor recruiting three different transcriptional repressers. Aim 3 We will determine how ERK switches the activity of LIN-1. These studies will elucidate the mechanisms and logic of a switch that is critical for cell fate determination during development. The results of these studies will significantly advance the understanding of how ETS transcription factors are regulated, how SUMO mediates transcriptional repression, and how ERK promotes cell proliferation, and address critical gaps in our current knowledge of these important areas. This switch may also respond to oncogenic signaling pathways, and these studies will provide information that can result in new therapeutic strategies for blocking the proliferation of tumor cells in humans. Lay language This research will reveal how tumor cells proliferate and suggest new strategies for treating human cancer
Keywords: enzyme substrate, enzyme substrate complex, gene induction /repression, mitogen activated protein kinase, transcription factor JUN kinase, binding site, biological signal transduction, developmental genetics, guanine nucleotide binding protein, phosphoprotein, phosphorylation, protein tyrosine kinase Caenorhabditis elegans, site directed mutagenesis
Project start date: 2000-01-01
Project end date: 2011-07-31
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MAP Kinase Regulation Of Substrate Proteins
Kerry Kornfeld, Associate Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899
Grant 5R01CA084271-07 from National Cancer Institute, IRG: CSD
Abstract: The Ras - ERK MAP kinase signaling pathway is critical for establishing cell fates during development. Importantly, mutations that activate the Ras pathway are a prevalent cause of human tumors. A major gap in our current understanding of this pathway is how activation of ERK alters the balance in gene expression to promote cell proliferation. Our long term goal is to understand how the Ras signaling pathway switches the activity of transcription factors so that they promote cell proliferation. We will address this by analyzing transcription factors that respond to Ras-mediated signaling in C. elegans and vertebrates. Understanding how the Ras pathway controls gene expression in development and disease in an important objective of medical research, since the information may lead to new and effective therapies for tumors caused by activated Ras signaling. Our preliminary results have defined exciting new mechanisms for the regulation of the LIN-1 transcription factor by Ras-mediated signaling and support two innovative hypotheses. (1) The LIN-1 transcription factor is post translationally modified by SUMO. Sumoylated LIN-1 represses transcription by interacting with MEP-1, DAS-1 and MAS-1, proteins involved in chromatin remodeling. (2) Activated ERK switches LIN-1 from a sumoylated, transcriptional represser that inhibits cell proliferation to a phosphorylated, transcriptional activator that induces cell proliferation. To test these hypotheses, we propose three specific aims. Aims 1 and 2 We will use biochemical techniques to define domains and specific residues of MEP-1, MAS-1 and DAS-1 that interact with LIN-1. We will characterize the relevance and specific functions of these interactions in intact animals. These studies will define the mechanisms of SUMO-mediated transcriptional repression and the significance of one DNA binding transcription factor recruiting three different transcriptional repressers. Aim 3 We will determine how ERK switches the activity of LIN-1. These studies will elucidate the mechanisms and logic of a switch that is critical for cell fate determination during development. The results of these studies will significantly advance the understanding of how ETS transcription factors are regulated, how SUMO mediates transcriptional repression, and how ERK promotes cell proliferation, and address critical gaps in our current knowledge of these important areas. This switch may also respond to oncogenic signaling pathways, and these studies will provide information that can result in new therapeutic strategies for blocking the proliferation of tumor cells in humans. Lay language This research will reveal how tumor cells proliferate and suggest new strategies for treating human cancer.
Keywords: enzyme substrate, enzyme substrate complex, gene induction /repression, mitogen activated protein kinase, transcription factor, JUN kinase, binding site, biological signal transduction, developmental genetics, guanine nucleotide binding protein, phosphoprotein, phosphorylation, protein tyrosine kinase, Caenorhabditis elegans, site directed mutagenesis
Project start date: 2000-01-01
Project end date: 2011-07-31
Grants awarded to Kerry Kornfeld
MAP Kinase Regulation Of Substrate Proteins
Kerry Kornfeld, Associate Professor
Developmental Biologywashington University
Grant 5R01CA084271-08 from National Cancer Institute, IRG: CSD
Abstract: The Ras - ERK MAP kinase signaling pathway is critical for establishing cell fates during development. Importantly, mutations that activate the Ras pathway are a prevalent cause of human tumors. A major gap in our current understanding of this pathway is how activation of ERK alters the balance in gene expression to promote cell proliferation. Our long term goal is to understand how the Ras signaling pathway switches the activity of transcription factors so that they promote cell proliferation. We will address this by analyzing transcription factors that respond to Ras-mediated signaling in C. elegans and vertebrates. Understanding how the Ras pathway controls gene expression in development and disease in an important objective of medical research, since the information may lead to new and effective therapies for tumors caused by activated Ras signaling. Our preliminary results have defined exciting new mechanisms for the regulation of the LIN-1 transcription factor by Ras-mediated signaling and support two innovative hypotheses. (1) The LIN-1 transcription factor is post translationally modified by SUMO. Sumoylated LIN-1 represses transcription by interacting with MEP-1, DAS-1 and MAS-1, proteins involved in chromatin remodeling. (2) Activated ERK switches LIN-1 from a sumoylated, transcriptional represser that inhibits cell proliferation to a phosphorylated, transcriptional activator that induces cell proliferation. To test these hypotheses, we propose three specific aims. Aims 1 and 2 We will use biochemical techniques to define domains and specific residues of MEP-1, MAS-1 and DAS-1 that interact with LIN-1. We will characterize the relevance and specific functions of these interactions in intact animals. These studies will define the mechanisms of SUMO-mediated transcriptional repression and the significance of one DNA binding transcription factor recruiting three different transcriptional repressers. Aim 3 We will determine how ERK switches the activity of LIN-1. These studies will elucidate the mechanisms and logic of a switch that is critical for cell fate determination during development. The results of these studies will significantly advance the understanding of how ETS transcription factors are regulated, how SUMO mediates transcriptional repression, and how ERK promotes cell proliferation, and address critical gaps in our current knowledge of these important areas. This switch may also respond to oncogenic signaling pathways, and these studies will provide information that can result in new therapeutic strategies for blocking the proliferation of tumor cells in humans. Lay language This research will reveal how tumor cells proliferate and suggest new strategies for treating human cancer
Keywords: enzyme substrate, enzyme substrate complex, gene induction /repression, mitogen activated protein kinase, transcription factor JUN kinase, binding site, biological signal transduction, developmental genetics, guanine nucleotide binding protein, phosphoprotein, phosphorylation, protein tyrosine kinase Caenorhabditis elegans, site directed mutagenesis
Project start date: 2000-01-01
Project end date: 2011-07-31
CDF-1 Regulation Of Zinc Homeostasis
Kerry Kornfeld, Associate Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899
Grant 5R01GM068598-04 from National Institute Of General Medical Sciences, IRG: BMT
Abstract: The long-term objective of this proposal is to characterize the mechanisms of zinc metabolism and the regulation of Zn2+-homeostasis in multicellular animals. Zn2+ metabolism has important implications for human health because zinc deficiencies caused by inadequate diet or inborn errors of metabolism result in many pathologies. Zn2+ also regulates important processes such as cell proliferation, and Zn2+ metabolism may affect diseases such as cancer. The first specific aim is to characterize the role of the C. elegans cdf-1 gene in Zn2+ metabolism in an intact animal. cdf-1 encodes a cation diffusion facilitator protein that promotes Zn2+ efflux across the plasma membrane. The research design and methods include developing assays of zinc content, distribution, uptake and excretion in nematode worms. These assays will be used to determine how changes in dietary zinc affect Zn2+-homeostasis. The role of cdf-1 in Zn2+ metabolism will be determined by analyzing mutants that lack CDF-1 activity, overexpress CDF-1, or express CDF-1 in specific tissues. The regulation of CDF-1 activity will be characterized by analyzing cdf-1 mRNA and protein in intact animals and determining how these products are regulated by dietary zinC. The final part of the first specific aim is to characterize the biochemical mechanism of action of CDF-1 in promoting Zn2+ transport across the plasma membrane. The research design and methods include developing assays of Zn2+ transport using purified components or cellular systems and characterizing the role of CDF-1 in Zn 2+ transport. Proteins that bind to CDF-1 will be identified using the yeast two-hybrid system and the role of CDF-l-interacting proteins in Zn2+ transport will be analyzed. The role of CDF-1-interacting proteins in intact animals will be investigated using genetic approaches. The second specific aim is to identify a network of genes that regulate Zn2+- homeostasis. The research design and methods include conducting genetic screens for mutations that affect C. elegans Zn2+ metabolism. Genetic methods will be used to determine the specific role of newly identified genes in Zn2+ metabolism. The role of these genes in cell fate specification will be determined to characterize the relationship between Zn2+ metabolism and Ras-mediated signaling. Molecular approaches will be used to clone the affected genes and reveal the mechanisms used by these proteins to regulate Zn2+ metabolism. These studies are likely to provide significant new insights into Zn2+ metabolism by establishing the role of CDF-1 in an intact animal, elucidating the biochemical mechanism of CDF-1, and identifying and characterizing new proteins that regulate Zn2+.
Keywords: homeostasis, metal metabolism, transcription factor, zinc, biological transport, dietary trace element, genetic regulation, messenger RNA, protein binding, protein kinase, Caenorhabditis elegans, atomic absorption spectrometry, fluorescence microscopy, genetic screening, mass spectrometry, molecular cloning, yeast two hybrid system
Project start date: 2003-06-01
Project end date: 2008-05-31
Identification Of Drugs That Delay Aging
Kerry Kornfeld, Associate Professor
Developmental Biologywashington University
1 Brookings Dr, Campus Box 1054
saint Louis, Mo 631304899
Grant 5R01AG026561-03 from National Institute On Aging, IRG: CMAD
Abstract: Aging is characterized by progressive, degenerative changes in many organ systems. These changes result in significant functional declines in elderly people and often contribute to death. Treatments that delay age- related degeneration would be desirable, but few are available. The long-term objective of this proposal is to identify drugs that can delay age-related degeneration. This proposal exploits nematode worms and mice as model systems to analyze aging. Nematodes are a powerful experimental system that is convenient for genetic, molecular, and pharmacologic studies, whereas mice are highly relevant to humans. The preliminary studies describe the identification of FDA-approved drugs that can extend the adult lifespan of worms including ethosuximide, trimethadione and valproic acid. Specific aim 1 proposes to characterize the mechanism of action of valproic acid by testing the hypothesis that valproic acid functions as an inhibitor of histone deacetylases. In addition, the hypothesis that ethosuximide and trimethadione function by modulating neural activity will be tested by analyzing mutants that are resistant to these drugs. A detailed, mechanistic understanding of the action of these drugs is important because it will elucidate endogenous pathways that influence aging, and establish a foundation for considering the therapeutic use of these drugs. Specific aim 2 proposes to identify FDA-approved drugs that can extend worm lifespan and characterize the mechanism of action of these drugs. The identification of drugs that delay aging is significant for two reasons. First, it might lead to the identification of new mechanisms that influence aging. Second, because these drugs are approved for human use, these studies might lead to therapies that can delay age-related degenerative changes in humans. Specific aim 3 proposes to determine if trimethadione and valproic acid can extend the lifespan of mice or delay age-related changes. The demonstration that these FDA-approved drugs delay aging in a vertebrate would be significant
Keywords: aging Chordata, acid, amidohydrolase, anticonvulsant, cataract, death, emotion, human, larva, lead, model, molecular genetics, mutant, organ, pharmacokinetics, pyrrolidinedione, stress, therapy
Project start date: 2006-08-15
Project end date: 2011-04-30
Related Publications
The anticonvulsant ethosuximide disrupts sensory function to extend C. elegans lifespan. PLoS Genet. 2008 Oct; 4( 10): e1000230. Epub 2008 Oct 24. PMID: 18949032 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. Genetics. 2008 Jun; 179( 2): 811-28. Epub 2008 May 27. PMID: 18505880 
The measurement and analysis of age-related changes in Caenorhabditis elegans. WormBook. 2008 Jan 24: 1-21. Review. PMID: 18381800 
Valproic acid extends Caenorhabditis elegans lifespan. Aging Cell. 2008 Jun; 7( 3): 305-17. Epub 2008 Jan 29. PMID: 18248662 Genetic and pharmacological factors that influence reproductive aging in nematodes. PLoS Genet. 2007 Feb 16; 3( 2): e25. Epub 2006 Dec 28. PMID: 17305431 Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp Gerontol. 2006 Oct; 41( 10): 1032-9. Epub 2006 Jul 26. Review. PMID: 16872777 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. Dev Biol. 2006 Sep 15; 297( 2): 550-65. Epub 2006 May 19. PMID: 16782085 Effects of anticonvulsant drugs on life span. Arch Neurol. 2006 Apr; 63( 4): 491-6. PMID: 16606760 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. 2006 Feb; 172( 2): 929-42. Epub 2005 Oct 11. PMID: 16219793 Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates. Development. 2005 Mar; 132( 5): 1047-56. Epub 2005 Feb 2. PMID: 15689373 Anticonvulsant medications extend worm life-span. Science. 2005 Jan 14; 307( 5707): 258-62. PMID: 15653505 Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates. Genetics. 2004 Aug; 167( 4): 1697-709. PMID: 15342509 Measurements of age-related changes of physiological processes that predict lifespan of Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2004 May 25; 101( 21): 8084-9. Epub 2004 May 12. PMID: 15141086 Identification of residues and domains of Raf important for function in vivo and in vitro. J Biol Chem. 2003 Nov 14; 278( 46): 45519-27. Epub 2003 Sep 3. PMID: 12954633 Zinc ions and cation diffusion facilitator proteins regulate Ras-mediated signaling. Dev Cell. 2002 May; 2( 5): 567-78. PMID: 12015965 Caenorhabditis elegans lin-45 raf is essential for larval viability, fertility and the induction of vulval cell fates. Genetics. 2002 Feb; 160( 2): 481-92. PMID: 11861555