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.