RECEPTOR INTERACTION WITH GTP-REGULATORY PROTEINS

Summary

Principal Investigator: Gary Johnson
Affiliation: University of North Carolina
Country: USA
Abstract: Many human diseases result from improper regulation of signaling networks in cells controlled by receptors and GTPases. The focus for this proposal is to define the role of the MAPK signaling network in controlling integrated physiological responses. MAPKs are members of a three kinase cascade composed of the MAPK, MAP2K and MAP3K. MAP3Ks integrate the MAPK signaling network in response to GTPases with parallel signaling pathways for control of physiological responses. MEKK1, a MAP3K, is the only kinase within the network to encode a kinase domain and a RING domain with E3 ligase activity. MEKK1 is also the only kinase in all genomes to encode a SWIM-RING domain architecture for binding and ubiquitinating protein substrates. MEKK1 is both a kinase for phosphorylation of specific substrates and an E3 ligase with a unique SWIM-RING domain architecture for ubiquitination of specific substrates. MEKK1 regulates the composition of the AP-1 transcription complex by regulating the levels of Fra-2 and JunB. MEKK1 is also capable of targeting c-Jun for ubiquitination and degradation. Our hypothesis is that MEKK1 functions to stimulate the activity of specific MAPK pathways (primarily JNK and ERK1/2), coordinately regulate the degradation of specific target substrates (e.g., c-Jun, JunB, Fra2), and controls AP-1 composition to alter specific gene expression for the integrated control of responses including migration, invasion, wound healing, inflammation, tissue remodeling and metastasis. The proposed studies are a systems approach to define how MEKK1 functions to control complex regulatory responses in cells and animals, which we believe will lead to novel therapeutic strategies for the treatment of diseases that involve tissue remodeling. Aim I will define function for the SWIM and RING domains of MEKK1 for binding and ubiquitination of target proteins. Approaches involve the use of quantitative mass spectrometry and ubiquitination assays to define protein substrates for the MEKK1 SWIM-RING domains. Aim II will provide genomic analysis of MEKK1-regulated gene expression using Chromatin-IP (ChIP) combined with promoter tiling arrays and single molecule-based DNA sequencing. The studies will define genes with differential binding of Fra2 and/or JunB in MEKK1-/- cells versus wild-type cells and the gene set whose expression is selectively altered by loss of MEKK1 regulation of AP-1 composition. Aim 3 will define physiological functions requiring E3 ligase activity and the kinase activity of MEKK1. The targeted knockout of MEKK1 generated in the last funding period provides a null background for add back of MEKK1 wild-type and mutant proteins. To this end, we have developed sensitive assays to measure MEKK1 function both in vitro and in vivo. Cumulatively, this work represents a mechanistic systems analysis of the function and regulation of an important MAP3K, MEKK1, which is critical in the control of homeostasis. MEKK1 has a unique position among the MAP3Ks and our discovery that MEKK1 regulates composition of AP-1 not just AP-1 activity, is a paradigm shift in how MAP3Ks regulate cellular physiology for the control of complex biological responses. PUBLIC HEALTH RELEVANCE: Dysregulation of the MAPK network is associated with many diseases including cancer, inflammation and metabolic disorders. Defining the global control not just of MAPK activity but protein degradation and gene expression by MEKK1 will identify novel therapeutic strategies for treating disease.
Funding Period: -------------------- - -------------------2
more information: NIH RePORT

Top Publications

  1. ncbi Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors
    A Beltran
    Department of Pharmacology and the Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7365, USA
    Oncogene 26:2791-8. 2007
  2. pmc MicroRNA 9-3p targets β1 integrin to sensitize claudin-low breast cancer cells to MEK inhibition
    Jon S Zawistowski
    Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC, USA
    Mol Cell Biol 33:2260-74. 2013
  3. pmc Coordination of Rho GTPase activities during cell protrusion
    Matthias Machacek
    Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, California 92037, USA
    Nature 461:99-103. 2009
  4. pmc Sequence patches on MAPK surfaces define protein-protein interactions
    Gary L Johnson
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 7365, USA
    Genome Biol 10:222. 2009
  5. pmc PB1 domain interaction of p62/sequestosome 1 and MEKK3 regulates NF-kappaB activation
    Kazuhiro Nakamura
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 7365, USA
    J Biol Chem 285:2077-89. 2010
  6. pmc The role of MEKK1 in hypertrophic cardiomyopathy
    John P Konhilas
    Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 0347, USA
    Int Heart J 51:277-84. 2010
  7. pmc MAP3K4/CBP-regulated H2B acetylation controls epithelial-mesenchymal transition in trophoblast stem cells
    Amy N Abell
    Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 7365, USA
    Cell Stem Cell 8:525-37. 2011
  8. pmc Efficiently identifying genome-wide changes with next-generation sequencing data
    Weichun Huang
    Biostatistics Branch, National Institute of Environmental Health Sciences, RTP, NC 27709, USA
    Nucleic Acids Res 39:e130. 2011
  9. pmc Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer
    Nicole Vincent Jordan
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA
    Cell Cycle 10:2865-73. 2011
  10. pmc Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer
    James S Duncan
    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
    Cell 149:307-21. 2012

Scientific Experts

  • Gary L Johnson
  • Weichun Huang
  • Amy N Abell
  • Kazuhiro Nakamura
  • Jon S Zawistowski
  • Nicole Vincent Jordan
  • Deborah A Granger
  • James S Duncan
  • John P Konhilas
  • Matthias Machacek
  • A Beltran
  • Joel S Parker
  • Brian T Golitz
  • Shawn M Gomez
  • David M Smalley
  • Pei Fen Kuan
  • Charles M Perou
  • H Shelton Earp
  • Xiaping He
  • Lee M Graves
  • Bing Zhou
  • Stephen V Frye
  • William Y Kim
  • Ben Major
  • David B Darr
  • Martin C Whittle
  • Jian Jin
  • Alicia A Midland
  • Xin Chen
  • Katherine A Hoadley
  • Norman E Sharpless
  • Jerry Usary
  • Nancy L Johnson
  • Leslie A Leinwand
  • Dana M Boucek
  • Todd R Horn
  • David P Siderovski
  • Adam J Kimple
  • Hunter Elliott
  • Perihan Nalbant
  • Louis Hodgson
  • Gaudenz Danuser
  • Olivier Pertz
  • Christopher Welch
  • Klaus M Hahn
  • B W Futscher
  • Y Liu
  • B D Cuevas
  • P Blancafort
  • S Parikh

Detail Information

Publications12

  1. ncbi Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors
    A Beltran
    Department of Pharmacology and the Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7365, USA
    Oncogene 26:2791-8. 2007
    ..Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors...
  2. pmc MicroRNA 9-3p targets β1 integrin to sensitize claudin-low breast cancer cells to MEK inhibition
    Jon S Zawistowski
    Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC, USA
    Mol Cell Biol 33:2260-74. 2013
    ..The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor...
  3. pmc Coordination of Rho GTPase activities during cell protrusion
    Matthias Machacek
    Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, California 92037, USA
    Nature 461:99-103. 2009
    ....
  4. pmc Sequence patches on MAPK surfaces define protein-protein interactions
    Gary L Johnson
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 7365, USA
    Genome Biol 10:222. 2009
    ..Recent studies on the modularity of mitogen-activated protein kinases show how redesigning 'surface patches' on a protein can change the topology of a signaling network...
  5. pmc PB1 domain interaction of p62/sequestosome 1 and MEKK3 regulates NF-kappaB activation
    Kazuhiro Nakamura
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 7365, USA
    J Biol Chem 285:2077-89. 2010
    ..The rear end acidic cluster of the p62 PB1 domain is used to organize cytosolic aggregates or speckles-associated TRAF6-p62-MEKK3 complex for control of NF-kappaB activation...
  6. pmc The role of MEKK1 in hypertrophic cardiomyopathy
    John P Konhilas
    Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 0347, USA
    Int Heart J 51:277-84. 2010
    ..We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis...
  7. pmc MAP3K4/CBP-regulated H2B acetylation controls epithelial-mesenchymal transition in trophoblast stem cells
    Amy N Abell
    Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 7365, USA
    Cell Stem Cell 8:525-37. 2011
    ..Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveals previously unrecognized genes potentially contributing to breast cancer...
  8. pmc Efficiently identifying genome-wide changes with next-generation sequencing data
    Weichun Huang
    Biostatistics Branch, National Institute of Environmental Health Sciences, RTP, NC 27709, USA
    Nucleic Acids Res 39:e130. 2011
    ..In particular, we show that our novel and robust 'parsimony' normalization method is superior to the widely-used 'tagRatio' method. Our software EpiCenter is freely available to the public...
  9. pmc Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer
    Nicole Vincent Jordan
    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA
    Cell Cycle 10:2865-73. 2011
    ..This intersection between EMT and stemness in TS cells and claudin-low metastatic breast cancer demonstrates the usefulness of developmental EMT systems to understand EMT in cancer...
  10. pmc Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer
    James S Duncan
    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
    Cell 149:307-21. 2012
    ..This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer...
  11. doi Defining MAPK interactomes
    Gary L Johnson
    Department of Pharmacology, University of North Carolina, Chapel Hill, 27599, United States
    ACS Chem Biol 6:18-20. 2011
    ..This discovery process provides a rich data and reagent resource for defining complexities of protein networks involving MAPKs and control of cellular physiology...