Figure Legends:
Figure 1: NF1 affects multiple signaling pathways to modulate Ras which can be therapeutically targeted through these various pathways. Left side exhibits NF1 dimerization and regulation through the proteasome. Specific NF1 variants might benefit from ubiquitin proteasome pathway (UPP) inhibitors to prevent excess degradation. Right side illustrates the Ras signaling pathway and the use of MEK inhibitors to slow tumor growth resulting from hyperactive Ras signaling.
Figure 2: Nf1 assay validations with WT mouse cDNA titrations in NF1 null HEK293 cells. WT NF1 +/+ HEK293 (293 +/+) cells were used as controls. Varying amounts (0-1000ng) of WT plasmid cDNA was transfected into the null cell line. A. Top panel – Quantitation of neurofibromin levels (n = 3). Bottom panel – Representative western blot of titration experiments. B. GTP-Ras activity levels as determined by Ras-GLISA assay (n = 3). Statistically significant differences were seen at 15ng and greater (red asterisk). C. Top panel – Quantitation of pERK/ERK levels (n = 3). Statistically significant differences were observed at 250ng. Bottom panel – Representative western blot. Error bars represent SEM and red asterisks indicate statistical significance.
Figure 3: Schematic of NF1 protein and putative domains with cDNA variants depicted. Red circles – nonsense mutations, blue squares – missense mutations, green triangles – in-frame deletion, and unfilled black circles – non-pathogenic variants. Amino acid positions are notated above protein domains in black text.
Figure 4: Relative neurofibromin protein levels for each cDNA.A. Quantitation of NF1 normalized to tubulin for all the Nf1cDNAs and categorized based on variant type. WT/tubulin ratios were set at 1.0 in each experiment with all other variant ratios relative to WT levels. Graph represents an n > 3 independent experiments for each cDNA. SEM error bars are shown. B. Representative western blot of select missense mutations showing NF1 and tubulin expression. C. Representative western blot of nonsense mutations showing NF1 and tubulin levels. Note the truncation products run at anticipated sizes.
Figure 5: Ras signaling activity assessed via GTP-Ras and pERK/ERK levels for each cDNA. A. Quantitation of GTP-Ras levels (blue bars) and pERK/ERK ratios (black bars) for all cDNAs. EV levels were set 1.0 and each sample was normalized and reported relative to EV levels. Comparisons to WT and statistical significance were determined via a Student’s t-test to determine if the variant negatively impacted NF1’s ability to repress Ras signaling. Blue asterisks indicate statistically significant impairment in the ability to inhibit GTP-Ras (p < 0.05). Black asterisks indicate statistically significant impaired ability to inhibit pERK/ERK ratios (p < 0.05). Graph represents N>3 independent experiments for each cDNA. Bars represent SEM. B. Representative image of one western blot of select missense mutations showing pERK and ERK levels. C. Representative image of one western blot of nonsense mutations showing pERK and ERK levels.
Figure 6: Analysis of protein stability and function to groupNf1 variants. Nf1 WT protein concentrations and corresponding GTP-Ras levels were plotted (gray dots) to generate a trend line (blue line). NF1 and GTP-Ras levels for each MS variant were overlaid onto the plot and variants were grouped as controls (green dots), splice (yellow dots), mild (orange dots), severe (pink dots), and unknown (teal dots). Clustering, based on NF1 stability and function is indicated by ovals: control variants – green oval, mild and splice variants – orange oval, select severe variants located in the GRD domain and interacting directly with Ras – pink oval, variants hugging the trend line- blue oval.