Fig. 2: mTORC1 phosphorylates APC/C during cell cycle entry.
From: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry
a, Schematic showing how mitogen-activated kinases may transiently inactivate APC/C. b,c, APC/C inactivation during cell cycle entry. b, The fraction of cells. Data are mean ± s.e.m. n = 5, 6, 5 and 3 independent experiments. c, Single-cell biosensor traces. n = 200 cells per condition. MCF-10A cells were starved for 48 h, then stimulated with mitogens with or without kinase inhibitors. P values were calculated using one-way ANOVA; NS, not significant. d, Immunoprecipitation analysis of CDH1 from MCF-10A cells (48 h starvation, 4 h mitogen stimulation) probed for the indicated proteins. Representative blot. n = 3. e, Co-immunoprecipitation of purified His–Sumo–CDH1 with DDK/Flag–mTOR or DDK/Flag–DYRK4 in a cell-free system. Representative blot. n = 3. f, In vitro kinase assay of purified CDH1 with recombinant mTOR complex, probed with a pan-phospho-serine/threonine/tyrosine antibody. Representative blot. n = 3. g, In vitro binding of CDH1 WT, T129A and T129D (200 ng each) to recombinant DDK–mTOR. Left, representative blot. Right, quantification. Data are mean ± s.d. n = 4. P values were calculated using one-way ANOVA. h, In vitro kinase assay of CDH1 mutants with recombinant DDK–mTOR, probed with a pan-phospho-threonine antibody. Representative blot. n = 3. i, Quantification of h. P values were calculated using one-way ANOVA. j, Immunoprecipitation of HA–CDH1 WT or T129A from MCF-10A cells (48 h starvation, 4 h mitogen stimulation), probed for the indicated proteins. Representative blot. n = 3. k, In vitro ubiquitylation reactions using recombinant APC/C, UBE2C and CDH1 WT, CDH1(T129A) or CDH1(T129D), plus recombinant mTOR and mLST8. Ubiquitinated cyclin BNTD was detected by fluorescence scanning. Representative gel. n = 3. l, Quantification of relative cyclin BNTD ubiquitination from k. Data are mean ± s.e.m. n = 3. P values were calculated using one-way ANOVA. m, Quantification of the percentage of the population with transient APC/C inactivation normalized to the control from Extended Data Fig. 5m. Data are mean ± s.d. from n = 4 (bars 1–4) or n = 3 (bar 5) independent experiments. P values were calculated using one-way ANOVA.
