Figure 2

Results of the functional assays on the TSC2 variants identified in family 1 (variants I820del and R1772C) and family 2 (variants T993M and L1511H). (A) Interaction between TSC1 and TSC2 variants. TSC1–TSC2 complexes were immunoprecipitated with antibodies specific for exogenous TSC1 from HEK 293 cells over-expressing TSC1 and wild-type TSC2 (wt) or TSC1 and the TSC2 variants. (B) Interaction between TSC1 and TSC2 variants. Ratio of coimmunoprecipitated TSC2:TSC1, as detected by immunoblotting, was estimated by densitometry scanning (Total Scan). (C) In vitro rheb GAP activity of immunoprecipitated TSC1–TSC2 complexes. Rheb-bound GDP/GTP ratios were determined after 90 minutes incubation with the immunoprecipitated wild-type TSC1–TSC2 complex (wt), protein A beads only (control), TSC1 only, or TSC1–TSC2 variant complexes. Rheb-bound GDP/GTP prior to addition of the TSC1–TSC2 complexes is shown (t = 0). (D) Inhibition of S6 phosphorylation in Tsc2 -/- MEFs. Percentage of Tsc2 -/- MEFs transfected with expression constructs encoding TSC1 and wild-type TSC2, or TSC1 and the TSC2 variants, and showing inhibition of S6 phosphorylation. Data from at least 3 separate experiments are shown. (E) TSC2-dependent inhibition of S6K-T389 phosphorylation. S6K, TSC1 and wild-type TSC2 (wt), or S6K, TSC1 and the TSC2 variants, were coexpressed in HEK 293 cells. Phosphorylation of S6K at the T389 position was determined by immunoblotting. A representative example of at least 3 separate experiments is shown. (E) TSC2-dependent inhibition of S6K-T389 phosphorylation. Ratio of total S6K:T389-phosphorylated S6K, as detected by immunoblotting, was estimated by densitometry scanning (Total Scan). Mean ratios relative to TSC2 wild-type (wt) are shown.