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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]
+ Show Author Affiliations
  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. doi:10.1074/jbc.ra119.010934.
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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
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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.
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@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}
}
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https://doi.org/10.1074/jbc.ra119.010934
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