skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Abstract

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in humanmore » cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.« less

Authors:
ORCiD logo [1];  [2]; ORCiD logo [3];  [1];  [1];  [1];  [1];  [4]; ORCiD logo [4]; ORCiD logo [4]; ORCiD logo [4];  [5];  [6];  [1];  [1];  [7]; ORCiD logo [1]
  1. Frederick National Lab. for Cancer Research, MD (United States)
  2. Univ. of California, San Francisco, CA (United States); Seoul National Univ. Hospital (South Korea)
  3. National Inst. of Health (NIH), Bethesda, MD (United States)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  5. Argonne National Lab. (ANL), Argonne, IL (United States)
  6. Frederick National Lab. for Cancer Research, MD (United States); Univ. of British Columbia, Vancouver, BC (Canada)
  7. Frederick National Lab. for Cancer Research, MD (United States); Univ. of California, San Francisco, CA (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)
OSTI Identifier:
1649499
Alternate Identifier(s):
OSTI ID: 1677648
Grant/Contract Number:  
AC05-00OR22725; HHSN261200800001E; AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 295; Journal Issue: 4; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; GTPase-activating protein (GAP); dimerization; Ras protein; GTPase Kras (KRAS); cell signaling; cancer; mitgen-activated protein kinase pathway; neurofibromin; NF1; mitogen-activated protein kinase pathway

Citation Formats

Sherekar, Mukul, Han, Sae-Won, Ghirlando, Rodolfo, Messing, Simon, Drew, Matthew, Rabara, Dana, Waybright, Timothy, Juneja, Puneet, O'Neill, Hugh, Stanley, Christopher B., Bhowmik, Debsindhu, Ramanathan, Arvind, Subramaniam, Sriram, Nissley, Dwight V., Gillette, William, McCormick, Frank, and Esposito, Dominic. Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer. United States: N. p., 2019. Web. https://doi.org/10.1074/jbc.ra119.010934.
Sherekar, Mukul, Han, Sae-Won, Ghirlando, Rodolfo, Messing, Simon, Drew, Matthew, Rabara, Dana, Waybright, Timothy, Juneja, Puneet, O'Neill, Hugh, Stanley, Christopher B., Bhowmik, Debsindhu, Ramanathan, Arvind, Subramaniam, Sriram, Nissley, Dwight V., Gillette, William, McCormick, Frank, & Esposito, Dominic. Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer. United States. https://doi.org/10.1074/jbc.ra119.010934
Sherekar, Mukul, Han, Sae-Won, Ghirlando, Rodolfo, Messing, Simon, Drew, Matthew, Rabara, Dana, Waybright, Timothy, Juneja, Puneet, O'Neill, Hugh, Stanley, Christopher B., Bhowmik, Debsindhu, Ramanathan, Arvind, Subramaniam, Sriram, Nissley, Dwight V., Gillette, William, McCormick, Frank, and Esposito, Dominic. Fri . "Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer". United States. https://doi.org/10.1074/jbc.ra119.010934. https://www.osti.gov/servlets/purl/1649499.
@article{osti_1649499,
title = {Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer},
author = {Sherekar, Mukul and Han, Sae-Won and Ghirlando, Rodolfo and Messing, Simon and Drew, Matthew and Rabara, Dana and Waybright, Timothy and Juneja, Puneet and O'Neill, Hugh and Stanley, Christopher B. and Bhowmik, Debsindhu and Ramanathan, Arvind and Subramaniam, Sriram and Nissley, Dwight V. and Gillette, William and McCormick, Frank and Esposito, Dominic},
abstractNote = {Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.},
doi = {10.1074/jbc.ra119.010934},
journal = {Journal of Biological Chemistry},
number = 4,
volume = 295,
place = {United States},
year = {2019},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Solubility survey of fragments of the neurofibromatosis type 1 protein neurofibromin
journal, May 2009

  • Bonneau, Fabien; Lenherr, Esther D.; Pena, Vladimir
  • Protein Expression and Purification, Vol. 65, Issue 1
  • DOI: 10.1016/j.pep.2008.12.001

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules
journal, May 1993

  • Gregory, Paula E.; Gutmann, David H.; Mitchell, Anna
  • Somatic Cell and Molecular Genetics, Vol. 19, Issue 3
  • DOI: 10.1007/BF01233074

Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s)
journal, July 2005


Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas
journal, July 2015

  • Krauthammer, Michael; Kong, Yong; Bacchiocchi, Antonella
  • Nature Genetics, Vol. 47, Issue 9
  • DOI: 10.1038/ng.3361

Mantid—Data analysis and visualization package for neutron scattering and μ SR experiments
journal, November 2014

  • Arnold, O.; Bilheux, J. C.; Borreguero, J. M.
  • Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Vol. 764
  • DOI: 10.1016/j.nima.2014.07.029

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ
journal, October 2016

  • Dharmaiah, Srisathiyanarayanan; Bindu, Lakshman; Tran, Timothy H.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 44
  • DOI: 10.1073/pnas.1615316113

EMAN2: An extensible image processing suite for electron microscopy
journal, January 2007

  • Tang, Guang; Peng, Liwei; Baldwin, Philip R.
  • Journal of Structural Biology, Vol. 157, Issue 1
  • DOI: 10.1016/j.jsb.2006.05.009

Restoring functional neurofibromin by protein transduction
journal, April 2018


Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies
journal, January 2019


Absolute calibration of small-angle neutron scattering data
journal, February 1987


Neurofibromin binds to caveolin-1 and regulates ras, FAK, and Akt
journal, February 2006

  • Boyanapalli, Madanamohan; Lahoud, Oscar B.; Messiaen, Ludwine
  • Biochemical and Biophysical Research Communications, Vol. 340, Issue 4
  • DOI: 10.1016/j.bbrc.2005.12.129

The NF1 gene in tumor syndromes and melanoma
journal, January 2017


Structural analysis of the GAP-related domain from neurofibromin and its implications
journal, August 1998


ATSAS 2.8 : a comprehensive data analysis suite for small-angle scattering from macromolecular solutions
journal, June 2017

  • Franke, D.; Petoukhov, M. V.; Konarev, P. V.
  • Journal of Applied Crystallography, Vol. 50, Issue 4
  • DOI: 10.1107/S1600576717007786

HEAT repeats – versatile arrays of amphiphilic helices working in crowded environments?
journal, October 2016


The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis
journal, August 2002

  • Hakimi, Mohamed-Ali; Speicher, David W.; Shiekhattar, Ramin
  • Journal of Biological Chemistry, Vol. 277, Issue 40
  • DOI: 10.1074/jbc.C200434200

Engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells
journal, November 2016


High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
journal, January 2017

  • Moriya, Toshio; Saur, Michael; Stabrin, Markus
  • Journal of Visualized Experiments, Issue 123
  • DOI: 10.3791/55448

Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation
journal, February 2013

  • Zhao, Huaying; Brautigam, Chad A.; Ghirlando, Rodolfo
  • Current Protocols in Protein Science, Vol. 71, Issue 1
  • DOI: 10.1002/0471140864.ps2012s71

KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner
journal, March 2019


Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase
journal, March 1996


mTORC2 facilitates endothelial cell senescence by suppressing Nrf2 expression via the Akt/GSK-3β/C/EBPα signaling pathway
journal, July 2018


MULCh : modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies
journal, January 2008

  • Whitten, Andrew E.; Cai, Shuzhi; Trewhella, Jill
  • Journal of Applied Crystallography, Vol. 41, Issue 1
  • DOI: 10.1107/S0021889807055136

Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients
journal, July 1990


Clinical and Molecular Characteristics of NF1-Mutant Lung Cancer
journal, February 2016


The NF1 somatic mutational landscape in sporadic human cancers
journal, June 2017


Structural and biochemical consequences of NF1 associated nontruncating mutations in the Sec14-PH module of neurofibromin
journal, January 2011

  • Welti, Stefan; Kühn, Sonja; D'Angelo, Igor
  • Human Mutation, Vol. 32, Issue 2
  • DOI: 10.1002/humu.21405

NF1 mutations are recurrent in adult acute myeloid leukemia and confer poor outcome
journal, June 2018


Elucidating the impact of neurofibromatosis-1 germline mutations on neurofibromin function and dopamine-based learning
journal, March 2015

  • Anastasaki, C.; Woo, A. S.; Messiaen, L. M.
  • Human Molecular Genetics, Vol. 24, Issue 12
  • DOI: 10.1093/hmg/ddv103

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium
book, January 2008

  • Cole, James L.; Lary, Jeffrey W.; Moody, Thomas P.
  • Biophysical Tools for Biologists, Volume One: In Vitro Techniques, p. 143-179
  • DOI: 10.1016/S0091-679X(07)84006-4

The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory
journal, June 2014

  • Heller, William T.; Urban, Volker S.; Lynn, Gary W.
  • Journal of Applied Crystallography, Vol. 47, Issue 4
  • DOI: 10.1107/S1600576714011285

Neurofibromatosis Type 1 Protein and Amyloid Precursor Protein Interact in Normal Human Melanocytes and Colocalize with Melanosomes
journal, March 2006

  • De Schepper, Sofie; Boucneau, Joachim M. A.; Westbroek, Wendy
  • Journal of Investigative Dermatology, Vol. 126, Issue 3
  • DOI: 10.1038/sj.jid.5700087

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Accurate assessment of mass, models and resolution by small-angle scattering
journal, April 2013


Mutational and functional analysis of the neurofibromatosis type 1 ( NF1 ) gene
journal, December 1996

  • Upadhyaya, M.; Osborn, Michael J.; Maynard, Julie
  • Human Genetics, Vol. 99, Issue 1
  • DOI: 10.1007/s004390050317

Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners
journal, August 2019

  • Carnes, Rachel M.; Kesterson, Robert A.; Korf, Bruce R.
  • Genes, Vol. 10, Issue 9
  • DOI: 10.3390/genes10090650

A novel bipartite phospholipid‐binding module in the neurofibromatosis type 1 protein
journal, February 2006


Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation
journal, June 2013

  • Zhao, Huaying; Ghirlando, Rodolfo; Piszczek, Grzegorz
  • Analytical Biochemistry, Vol. 437, Issue 1
  • DOI: 10.1016/j.ab.2013.02.011

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21
journal, November 1990


A shared molecular mechanism underlies the human rasopathies Legius syndrome and Neurofibromatosis-1
journal, July 2012

  • Stowe, I. B.; Mercado, E. L.; Stowe, T. R.
  • Genes & Development, Vol. 26, Issue 13
  • DOI: 10.1101/gad.190876.112

The RasGAP Proteins Ira2 and Neurofibromin Are Negatively Regulated by Gpb1 in Yeast and ETEA in Humans
journal, February 2010

  • Phan, Vernon T.; Ding, Vivianne W.; Li, Fenglei
  • Molecular and Cellular Biology, Vol. 30, Issue 9
  • DOI: 10.1128/MCB.01450-08