NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702.
Helen Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158.
Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Republic of Korea.
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia 30322.
Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.
Argonne National Laboratory, Lemont, Illinois 60439.
Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702.
Department of Biochemistry, Life Sciences Center, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada.
NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702 dom.esposito@nih.gov.
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 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.
GTPase Kras (KRAS)GTPase-activating protein (GAP)NF1Ras proteincancercell signalingdimerizationmitogen-activated protein kinase pathwayneurofibromin
Figures
Figure 1.Full-length neurofibromin is a high-affinity dimer.A, SDS-PAGE analysis of purified full-length neurofibromin. Lanes are molecular mass markers (M) with the sizes of relevant bands shown in kDa, clarified extract from the insect cell expression (lane 1), elution pool from IMAC chromatography (lane 2), and purified protein after size-exclusion chromatography (lane 3). B, analytical SEC trace of purified neurofibromin (orange) compared with molecular weight standards (blue). Standards used were blue dextran (peak 1), thyroglobulin (peak 2, 669 kDa), ferritin (peak 3, 440 kDa), aldolase (peak 4, 158 kDa), conalbumin (peak 5, 75 kDa), and ovalbumin (peak 6, 43 kDa). C, SEC-MALS analysis of full-length neurofibromin. D, SAXS/SANS analysis of full-length neurofibromin. Red (1 mg/ml) and blue (0.5 mg/ml) lines are SAXS data from runs with two different concentrations of neurofibromin, and open circles represent SANS data using 1 mg/ml neurofibromin. E, sedimentation velocity absorbance c(s) profiles for NF1 at 0.6 μm (red) and 1.2 μm (blue) based on data collected at 280 nm. The inset shows the corresponding c(s) profile for NF1 at 25 nm (green) based on absorbance data collected at 230 nm. F, Western blotting of immunoprecipitation of differentially epitope-tagged NF1 proteins from HEK293 cells. The top two gel sections contain lysate purified with anti-FLAG antibodies. The bottom two sections contain WCL. In both cases, the samples are probed with antibodies to the FLAG or V5 epitopes as noted. Molecular mass of standards is noted on the right in kilodaltons.Figure 2.Negative-stain EM of full-length neurofibromin.A, representative transmission electron micrograph of full-length neurofibromin protein. Scale bar (100 nm) is shown for comparison. B, representative 2D class averages obtained from 41,951 particles. Each box shown is 50 × 50 nm. C, 20-Å density map of full-length neurofibromin generated from 294 class averages shown in multiple orientations. Proposed protomers are indicated with green and blue coloring.Figure 3.Neurofibromin fragments and domain architecture. Schematic of the regions utilized for production of neurofibromin proteins in this study. Domains have amino acid boundaries as noted and are designated by letters A–F. Corresponding regions of interest identified from the literature are noted above. Abbreviations used are as follows: CSRD, cysteine–serine-rich domain; SBR, syndecan-binding region.Figure 4.N-terminal domains of neurofibromin are monomeric.A, SDS-polyacrylamide gel of purified ABC and ABCD proteins. Size of molecular mass markers are noted in kilodaltons. B, SEC-MALS analysis of ABC (blue) and ABCD (red) fragments. C, sedimentation velocity absorbance c(s) profiles for the ABC protein at 1.1 μm (green), 2.2 μm (red), and 4.5 μm (blue) based on data collected at 280 nm. D, sedimentation velocity absorbance c(s) profiles for the ABCD protein at 1.1 μm (green), 2.1 μm (red), and 4.2 μm (blue) based on data collected at 280 nm. E, Western blotting of immunoprecipitation of differentially epitope-tagged NF1 proteins in HEK293 cells. In this figure, HA-ABCD protein was co-IPed with FLAG-tagged domains as noted. The top two gel sections are lysates purified with anti-FLAG antibodies and probed with antibodies to the HA or FLAG epitopes. The bottom section contains WCL probed with anti-HA antibodies. Molecular mass of standards is noted on the right in kilodaltons. Circled regions are discussed in more detail in the text. EV, empty vector control; FL, full-length NF1.Figure 5.C-terminal domains of neurofibromin are capable of dimerization.A, SDS-polyacrylamide gel of purified CDEF, DEF, and EF proteins. Size of molecular mass markers are noted in kilodaltons. B, SEC-MALS analysis of CDEF (blue), DEF (red), and EF (green) proteins. C, sedimentation velocity absorbance c(s) profiles for the DEF protein at 2.1 μm (green), 4.2 μm (red), and 8.8 μm (blue) based on data collected at 280 nm. D, sedimentation velocity absorbance c(s) profiles for the CDEF protein at 1.2 μm (black), 2.6 μm (green), 5.2 μm (red), and 10.5 μm (blue) based on data collected at 280 nm. Data at the highest concentration was collected using a 3-mm pathlength cell, and standard 12-mm cells were used for the lower concentrations. E, Western blotting of co-immunoprecipitation of differentially epitope-tagged NF1 proteins in HEK293 cells. In this figure, HA-CDEF protein was co-IPed with FLAG-tagged domains as noted. The top two gel sections are lysates purified with anti-FLAG antibodies and probed with antibodies to the HA or FLAG epitopes. The bottom section contains WCL probed with anti-HA antibodies. Molecular mass of standards is noted on the right in kilodaltons. Circled regions are discussed in more detail in the text. EV, empty vector control; FL, full-length NF1.Figure 6.Reconstitution of full-length neurofibromin dimers from ABC and DEF proteins.A, SDS-PAGE analysis of SEC of a equimolar mixture of ABC and DEF proteins. Size of molecular mass markers in lane M are noted in kilodaltons. Lane L represents the loaded material, and additional lanes are elution fractions across the column. B, transmission electron micrograph of reconstituted neurofibromin dimers from the ABC/DEF mixture. Scale bar (100 nm) is noted. C, representative 2D class averages from particles selected from multiple transmission electron microscopic micrographs. Scale bar (30 nm) is noted. D, sedimentation velocity absorbance c(s) profiles for equimolar mixtures of the N-terminal ABC and C-terminal DEF fragments of NF1 at 1.0 μm (green), 2.0 μm (red), and 4.5 μm (blue) based on data collected at 280 nm. Data at the highest concentration were collected using a 3-mm pathlength cell; standard 12 mm cells were used for the lower concentrations. E, Western blotting of co-immunoprecipitation of differentially epitope-tagged NF1 proteins in HEK293 cells. In this figure, HA-DEF protein was co-IPed with FLAG-tagged domains as noted. The top two gel sections are lysates purified with anti-FLAG antibodies and probed with antibodies to the HA or FLAG epitopes. The bottom section contains WCL probed with anti-HA antibodies. Molecular mass of standards are indicated on the right in kilodaltons. Circled regions are discussed in more detail in the text. EV, empty vector control; FL, full-length NF1.Figure 7.Identification of the interaction between the neurofibromin C and EF domains. SDS-PAGE analysis of SEC of equimolar mixtures of NF1 domains with EF(1821–2818). In each panel, the size of molecular mass markers in lane M is noted in kilodaltons. Lane L represents the loaded material, and additional lanes are elution fractions across the column. Neurofibromin fragments mixed with the EF domain in each panel were as follows: A, 1085–1816; B, 1085–1530; C, 1541–1820.Figure 8.RAS GTPase stimulation by neurofibromin domains.A, phosphate sensor assay using purified neurofibromin proteins. The level of stimulation of basal RAS GTPase activity is shown for four different neurofibromin proteins and an equimolar mixture of ABC and DEF proteins. Data shown are the averages of two separate assays, each performed in triplicate, with standard deviations shown with error bars. B, RAS–GTP ELISA carried out in HEK293 cells lacking endogenous NF1. The plotted data are measurements of RAS–GTP levels after EGF stimulation of serum-starved cells that were transfected with various NF1 constructs. Data are percentages of a control experiment with GFP and are averages of three or four replicate wells for each sample. Standard deviations are noted with error bars.
그림 출처: PubMed Central 공개 논문 (PMC6983858)
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article
그 밖의 정보
출판 형태
Journal Article, Research Support, N.I.H., Extramural, Research Support, N.I.H., Intramural, Research Support, U.S. Gov't, Non-P.H.S.
저널
The Journal of biological chemistry · eISSN 1083-351X
