Extended Data Fig. 4: Human TSC2(S1364) or TSC2(S1365) mutated to glutamatic acid (S1364E or S1365E) suppresses ET1-stimulated cardiomyocyte hypertrophy and mTORC1 activation, whereas mutation to alanine (S1364A or S1365A) amplifies both.
From: PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress
a, Nppb mRNA expression (pathological hypertrophy gene marker) in rat cardiomyocytes transfected with human Flag–TSC2(WT), Flag–TSC2(S1365A) or Flag–TSC2(S1365E) and then exposed to 48 h ET1 (to induce hypertrophy) or to vehicle. Activation of PKG1 by sildenafil reduces ET1-stimulated Nppb in TSC2(WT)-expressing cells, but not in cells expressing TSC2(S1365A) or TSC2(S1365E) mutants. TSC2(S1365E) expression depresses the increase in Nppb expression with ET1 stimulation, whereas TSC2(S1365A) expression enhances it. These results are nearly identical to those shown in Fig. 2a in which the human TSC2(S1364) (first serine of the duplet) was mutated. This shows that genetic modulation of either serine results in the same biological modulation of ET1 stimulation on growth and mTORC1 activity. Data are mean ± s.d., n = 6 biologically independent experiments, one-way ANOVA with Tukey multiple comparisons test, *P < 1 × 10−6 versus other TSC2(WT) groups; †P = 0.002, ††P = 0.001 versus TSC2(S1365E), #P < 1 × 10−6 versus TSC2(S1365E) and TSC2(S1365A) and ET1, ‡P < 1 × 10−6 versus TSC2(S1365E), §P < 1 × 10−6 versus TSC2(S1365E) and TSC2(WT) + ET1 and sildenafil. b, Summary analysis of immunoblots displayed in Fig. 1b. Values are normalized to TSC2(WT) treated with vehicle; data are mean ± s.d., n = 4 (LC3-II) or 6 (others) biologically independent experiments; one-way ANOVA with Tukey multiple comparisons test, *P ≤ 7 × 10−6 versus vehicle control, †P < 1 × 10−6 versus TSC2(S1364A) and ET1, ‡P < 5 × 10−6, #P = 0.01 versus TSC2(WT) and ET1. c, Example immunoblots from the same experiment as in a, showing changes in mTORC1 signalling proteins, p62 and LC3-II. ET1 stimulates phosphorylation of mTORC1 targets (p70S6K, 4E-BP1 and ULK1) and increases LC3-II and p62—consistent with mTORC1 activation and enhanced autophagy. Human TSC2(S1365E) reduces mTORC1 activation and p62 and increases LC3-II, whereas human TSC2(S1365A) does the opposite. This is identical to responses found using human S1364A and S1364E mutants (b and Fig. 2b), confirming the functional equivalency of either serine modification. Experiments were replicated 2–4 times, n = 4–8 biologically independent samples. d, Summary data for this experiment. Values normalized to TSC2(WT) treated with vehicle; data are mean ± s.d., n = 8 independent replicates for p70S6K and 4E-BP1, n = 6 for ULK1 and n = 4 for p62 and LC3-II. One-way ANOVA with Tukey multiple comparisons test, Results of pairwise comparisons: *P < 1 × 10−6 versus corresponding TSC2 genotype and vehicle, †P ≤ 1 × 10−6 versus TSC2(WT) and ET1 and TSC2(S1365A) and ET1,#P = 0.003, ‡P = 0.0001, ¶P = 0.06, §P = 5 × 10−6, %P < 1 × 10−6 versus TSC2(WT) and ET1.
