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

Mukul Sherekar,Sae-Won Han,Rodolfo Ghirlando,Dominic Esposito,Puneet Juneja,Timothy Waybright,Sriram Subramaniam,Debsindhu Bhowmik,Dwight V Nissley,Simon Messing,Arvind Ramanathan,Dana Rabara,Frank Mccormick,Christopher B Stanley,William Gillette,Matthew Drew,Hugh O'Neill

doi:10.1074/jbc.ra119.010934

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.

Highlights

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I
The 2818 amino acid neurofibromin protein is involved in a variety of human diseases, including several sporadic cancers, as well as the common genetic disorder neurofibromatosis type
The underlying mechanism of these diseases seems to involve defects in the GTPase-activating activity of neurofibromin, which regulates the RAS family of small GTPases and downstream mitogen-activated protein kinase pathways, this activity resides in a small portion of the protein

Summary

Results

Purified full-length human neurofibromin produced in baculovirus-infected insect cells migrated on SDS-PAGE at a molar mass close to its predicted size of 317 kDa (Fig. 1A). Sedimentation experiments on CDEF at 8.8 ␮M showed the presence of a species at 11.65 S with an estimated molar mass of 300 kDa (Fig. 5D) These data suggest that the CDEF domain is dimeric and the smaller than expected mass can be attributed to the selfassociation observed as the concentration is raised from 2.2 to 8.8 ␮M. Using HEK293 cells lacking endogenous NF1, we were able to show that the CDEF and DEF domains of differentially epitope-tagged neurofibromin did co-IP (Fig. 5E, green circled region), confirming the potential for dimerization in cells similar to that observed with purified proteins. We observed similar reconstitution results using ABCD and EF proteins, and sedimentation studies of these mixtures in the 1.0 to 5.5 ␮M concentration range showed the presence of a species at 14.26 S with an estimated mass of 580 Ϯ 95 kDa (data not shown). The isolated GRD domain (1198 –1530) consistently showed higher levels of GAP activity compared with other NF1 domains, suggesting that some inhibition of activity in the larger domains may be

B ML 116

Discussion

Experimental procedures