Analysis of the TSC1 L117P variant. Wild-type TSC1 (wt) or the L117P, N198F199delinsI (del) or R692X variants (all containing a C-terminal myc epitope tag) were overexpressed in HEK 293T cells alone (a) or in the presence of coexpressed TSC2 (b). Cell lysates were separated into post 10 000 g centrifugation pellet and supernatant fractions. The TSC1 variants were detected with a monoclonal mouse antibody against the myc epitope tag (9B11; Cell Signaling Technology). TSC1–TSC2 complexes were immunoprecipitated from the supernatant fractions using the same antibody. TSC2 was detected with a polyclonal rabbit antiserum.⁸ (a) Expression of the TSC1 variants in the absence of TSC2. Wild-type TSC1 (wt) was detected predominantly in the pellet fraction. In contrast, the variants were detected predominantly in the supernatant fraction. The signals for the L117P and N198F199delinsI (del) variants are clearly less than wild-type TSC1 and the R692X variants (as detected with the 9B11 antibody). (b) Coexpression of TSC2 and the TSC1 variants. In the presence of TSC2, wild-type TSC1 and the variants were detected in both subcellular fractions. Coexpression of TSC2 resulted in a shift of wild-type TSC1 to the supernatant fraction and a shift of the variants to the pellet fraction. TSC1 and the TSC1 variants were immunoprecipitated with an antibody against the myc epitope tag. TSC2 was coimmunoprecipitated with all three variants. However, in each case, the TSC2 signal in the immunoprecipitate was clearly less than with the wild-type TSC1.

Results of the functional assays on the M224R, G1035S and M224R/G1035S variants. (a) TSC1–TSC2-dependent inhibition of S6K-T389 phosphorylation. TSC2, S6K and wild-type TSC1 (wt TSC1), or the TSC1 variants, were expressed in HEK 293T cells. T389 phosphorylation of S6K was determined by immunoblotting and was reduced in the presence of wild-type TSC1 and the G1035S variants only. S6K, wild-type TSC1 and the TSC1 variants were detected with an antibody against the myc epitope tag. The signal for the M224R and M224R/G1035S variants is reduced compared with wild-type TSC1 and the G1035S and R692X variants. A representative example of at least three separate experiments is shown. (b) Inhibition of S6 phosphorylation in Tsc1−/− MEFs. Wild-type myc-tagged TSC1 (TSC1) and the myc-tagged variants were expressed in Tsc1−/− MEFs. S6 S235/236 phosphorylation in the myc-positive cells was determined by double-label immunofluorescent microscopy using a rabbit polyclonal antibody specific for S235/236-phosphorylated S6 and a mouse monoclonal antibody specific for the myc tag. The percentage of Tsc1−/− MEFs expressing the TSC1 variants and showing a clear reduction in S6 phosphorylation is indicated. Because the signals for the M224R and M224R/G1035S variants were very low, it was difficult to unequivocally differentiate >50 expressing cells. Therefore, the total number of counted cells after two experiments is indicated above each bar.

Pedigrees of the investigated families with TSC. Arrows indicate the index cases. Open symbols indicate no signs or symptoms of TSC; black symbols indicate individuals with definite TSC; hatched symbols indicate individuals with possible TSC. A question mark indicates individuals where no clinical data were available. Genotypes are indicated for the individuals where DNA was available for testing. (a) Family with TSC and the TSC1 c.350T>C (p.L117P) variants. (b) Family with TSC and cosegregation of the TSC1 c.671T>G (p.M224R) and c.3103G>A (p.G1035S) variants. (c) Family with cosegregation of TSC and the TSC1 c.539T>C (p.L180P) variants. (d) Family with a de novo TSC1 c.572T>A (p.L191H) mutation.

TSC1–TSC2-dependent inhibition of mTOR signalling by the TSC1 L117P variant. (a) T389 phosphorylation of S6K in HEK 293T cells coexpressing TSC2, myc-tagged S6K and myc-tagged wild-type TSC1 (wt) or the TSC1 L117P, N198F199delinsI (del) or R692X variants was determined by immunoblotting. Cells transfected with differing amounts of wild-type or variant expression constructs, as indicated, were analysed. The first lane on the left (control) corresponds to cells transfected with S6K and wild-type TSC1 expression constructs only (no TSC2 expression construct). The coexpression of only TSC2 and wild-type TSC1 inhibited S6K phosphorylation. (b) TSC1-dependent inhibition of S6 phosphorylation. YFP-tagged wild-type TSC1 (YFPTSC1) and the YFP-tagged N198F199delinsI (YFPdel) and L117P (YFPL117P) variants were expressed in Tsc1−/− MEFs. S6 S235/236 phosphorylation in the YFP-positive cells was detected by immunofluorescent microscopy using an antibody specific for S235/236-phosphorylated S6 (Cell Signaling Technology). As a control, S6 phosphorylation in cells expressing YFP only was also determined. At least 50 cells were counted per variant per experiment. Mean percentage scores of three separate experiments are shown. Values significantly different from the wild type are indicated.

Analysis of the 128delV, L180P, L191H, R246K, G305R, G305W, R509Q and R1097H variants. (a) TSC1-dependent inhibition of S6K T389 phosphorylation. S6K T389 phosphorylation was determined by immunoblotting as before except that the blots were developed using Odyssey near infrared detection (Li-Cor Biosciences). Cells expressing S6K, TSC2 and wild-type TSC1 (wt) or the TSC1 variants were analysed. As controls, cells transfected with expression constructs for wild-type TSC1 and S6K only (TSC1/S6K), TSC2 and S6K only (TSC2/S6K), S6K only (S6K) or empty vector only (pcDNA3) were also analysed. S6K and the TSC1 variants were detected with an antibody specific for the myc epitope tag. The R692X truncation variant is indicated with a large arrowhead; molecular weight markers are indicated with small arrows. Expression of wild-type TSC1 or the R246K, G305R, G305W, R509Q, G1035S and R1097H variants clearly inhibited S6K phosphorylation. The signals for the L117P, 128delV, L180P, L191H and M224R variants were clearly reduced compared with wild-type TSC1 (see also panel b), and S6K T389 phosphorylation was increased (see also panel c). A representative example of three separate experiments is shown. (b) Relative expression of the TSC1 variants. The integrated intensities of the TSC1 and TSC2 signals for each variant were determined in three independent experiments using the Odyssey scanning software. The mean TSC2/TSC1 ratios, relative to the wild-type TSC1 (wt) (wild-type TSC1 TSC2/TSC1 ratio=1), were determined. Standard deviations are indicated. (c) TSC1-dependent inhibition of S6K T389 phosphorylation. The ratio of the T389 S6K phosphorylation signal intensity to the total S6K signal intensity (T389/S6K) was measured in three independent experiments. The integrated intensity of each band was determined using the Odyssey scanning software and the mean T389/S6K ratios, relative to the wild-type TSC1 (wt) (wild-type TSC1 T389/S6K ratio=1), were determined. Standard deviations are indicated. (d) Inhibition of S6 phosphorylation in Tsc1−/− MEFs. Wild-type myc-tagged TSC1 (wt) or the myc-tagged variants were expressed in Tsc1−/− MEFs. S6 S235/236 phosphorylation in the myc-positive cells was determined by immunofluorescent microscopy as before. At least 50 cells were counted per variant per experiment. Each variant was tested in at least two separate experiments. Mean percentages of Tsc1−/− MEFs expressing the TSC1 variants and showing reduced phosphorylation of S6 are shown. Standard deviations are indicated.