Effects of Smith-Kingsmore Syndrome associated MTOR variants on the mTOR signaling cascade
Creators: name: Jonathan Thulson
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2023

description: Introduction: Smith-Kingsmore Syndrome (SKS) is a rare autosomal dominant disorder that presents 
with varying levels of intellectual disability, developmental delay, megalocephaly, seizures, and large and 
dysmorphic neurons. SKS is a disorder caused by de novo variants in MTOR that lead to hyperactivation 
of the mTOR signaling pathway via activation of the mTORC1 and mTORC2 complexes. The extent to 
which pathogenic variants act through mTORC1 and mTORC2 complexes to upregulate mTOR signaling 
may dictate the variable clinical presentation and the extent to which patients may respond to rapamycin, 
a selective mTORC1 inhibitor. In this study we will evaluate 12 SKS-causing variants on mTORC1 and 
mTORC2-complex signaling to determine which variants may be rapamycin sensitivity. 

Methods: Wild-type (WT) and 12 mutant MTOR clones were overexpressed in a human embryonic 
kidney cell line (HEK-293T). Cells were lysed 48 hours after transfection and whole cell lysates were 
collected for immunoblotting techniques to probe for the desired downstream proteins. Phosphorylated 
and total protein abundance was determined for each of the variants and compared to WT to evaluate 
the extent to which SKS-variants activate mTORC1 (S6K and 4EBP1) and mTORC2 (AKT) complexes. 
Densitometry utilizing ImageJ was utilized to compare band densities. 

Results: In the preliminary analysis, several of the variants showed variable effects on mTORC1 and 
mTORC2 activation. The two markers of mTORC1 activation (p4EBP1 and pS6K) were highly correlated. 
Specifically, K1395R, F1888C, and D2412V did not have greater than 2-fold increase in phosphorylation 
abundance compared to WT for any of the downstream proteins. W1490R and I2501V had greater than 
2-fold increase in abundance for all of the different proteins. M1595I variant showed an increase in S6K
(5.17-fold) and 4EBP1 (9.75-fold), while the abundance for AKT (2.60-fold) was lower. The E1799K 
mutation had greater than 2-fold increase in S6K and 4EBP1 but not AKT. The C1483F and C1483Y 
variants showed this same trend. The F2202C variant had greater than 2-fold increase for 4EBP1 but not 
AKT or S6K; and the A1832T mutation had a greater 2-fold increase in S6K but not AKT or 4EBP1. A 
technical replicate repeating the immunoblotting on the same lysate revealed inconsistent results and 
specifically loss of signal associated with the phosphorylated markers which is likely explained by these 
protein products being labile. Secondary analysis of four variants (K1395R, C1483F, C1483Y, and 
E1799K) showed inconsistent results with the preliminary analysis. 

Conclusions: Variants M1595I, E1799K, C1483F, and C1483Y showed preliminary trends of selective 
activation of mTORC1. The other variants did not show trends of selective activation. Despite these initial 
trends, repeat analysis showed different patterns of mTORC1 and mTORC2 activation for each of the 
variants. Further optimization of immunohistochemistry techniques is warranted to acquire consistent 
results for true determination of selective mTOR complex activation.