Extended Data Fig. 3: PKG1 is activated by ET1 in rat cardiomyocytes and results in TSC2 phosphorylation detected by a human TSC2(S1364) antibody in cells expressing human TSC2(WT) but not human TSC2(S1364A) or TSC2(S1365A); this phosphorylation occurs by direct modification of TSC2 by PKG1α.
From: PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress
a, PKG1 activation in cardiomyocytes expressing human TSC2(WT), TSC2(S1365A) or TSC2(S1365E) and stimulated with ET1 (10 nM) versus vehicle for 48 h. Data are mean ± s.d., n = 18 biologically independent samples, two-tailed unpaired Student’s t-test. b, From same experiment, PKG1 activation is found to be independent of the form of TSC2 expressed; n = 6 biologically independent samples, box and whisker and raw data plots. Data normalized to median for wild type (without ET1); P = 0.0004 for ET1 effect, P > 0.8 for TSC2 genotype effect by two-way ANOVA. c, Summary data for phosphorylated/total human TSC2(S1364) from the rat cardiomyocyte ET1-stimulation experiment shown in Fig. 1f. Data are mean ± s.d., n = 6 biologically independent samples from three experiments, one-way ANOVA with Tukey multiple comparisons test, #P = 0.003 versus TSC2(S1365E), P = 0.0003 versus TSC2(S1365A). d, Antibody raised against TSC2(S1365) (mouse) (equivalent to human TSC2(S1364)) shows increased TSC2 phosphorylation in rat cardiomyocytes transfected with human TSC2(WT), but not cells expressing human TSC2(S1365A) or human TSC2(S1365E). Summary from three independent replicates yielding n = 6 biologically independent samples, data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, #P = 0.0002 versus TSC2(S1365E), P = 0.0007 versus TSC2(S1365A). The results are identical to those using mouse TSC2(S1365) (human TSC2(S1364)) mutants displayed in Fig. 1i, indicating that mutations at either serine (human sequence: S1364 or S1365; mouse sequence S1365 or 1366) prevents phosphorylation of the other and/or its detection by the phosphorylation-specific antibody. e, Direct TSC2 phosphorylation by recombinant PKG1α detected by autoradiography on human Flag–TSC2(WT) and Flag–TSC2(S1365A). Experiments were replicated three times (n = 6 biologically independent samples) with identical results. The result is identical to that in Fig. 1j with human HA–TSC2(S1364A). f, Direct TSC2 phosphorylation by PKG1α in lysates from TSC2 knockout HEK293 cells expressing human TSC2(WT) or human HA–TSC2(S1364A) or human Flag–TSC2(S1365A), PKG1α(M438G), and N6-benzyl-ATPγS, and probed for thiophosphate ester. Top, data with human TSC2(S1364) mutated; bottom, with human TSC2(S1365) mutated. The results are identical. n = 6 biologically independent samples for each assay.
