Fig 2: RNF167-mediated ubiquitination and AKT1-mediated phosphorylation of CASTOR1 promotes breast cancer progression.a, b Weaker suppression of colony formation of ER+ (a) and HER2+ (b) breast cancer cells in softagar by CASTOR1 S14D than WT and S14A. c CASTOR1 silencing enhanced colony formation in softagar of T47D cells. d–g Overexpression of CASTOR1 S14D had a less suppressive effect on breast tumor growth and a lower extended animal survival rate than WT and S14A had in a breast cancer xenograft model; the tumor volumes at the indicated time point post-inoculation were measured (d); the tumor volumes of the last time point were compared (e), and the actual tumors (f) and the survival rates (g) are shown. h–j CASTOR1 knockdown promoted tumor growth and shortened animal survival rate. The tumor volumes at the indicated time point post-inoculation were measured (h); and the tumor volumes of the last time point (i) and the survival rates (j) are shown. The lower panels of a–c were quantifications of colony numbers from three independent experiments presented as mean ± SEM and analyzed by one-way ANOVA. For d, e, g, each mouse group contains 20 tumors (n = 20). For h, j, the Vector control, shRNA1, and shRNA2 groups contain 15, 11, and 10 tumors, respectively (n = 15, 11, and 10). For i, the Vector control, shRNA1, and shRNA2 group contain 6, 7, and 5 tumors, respectively (n = 6, 7, and 5). For e, i, the boundary closest to the zero indicates the 25th percentile, a line within the box means the median, and the boundary of the box farthest from zero marks the 75th percentile. Whiskers (error bars) above and below the box indicate the minima and maxima. d, e, h, i were presented as mean ± SEM and analyzed by two-sided Student’s t test. g, j was analyzed by two-sided Log-rank test. “*” and “***” denote P < 0.05 and P < 0.001, respectively. Scale bars: 200 µM (a–c). Source data are provided in Source data file.

Fig 3: AKT-mediated phosphorylation and RNF167-mediated ubiquitination of CASTOR1 release mTORC1 inactivation by CASTOR1.a CASTOR1 bound to MIOS in a dose-dependent manner. b Overexpression of RNF167 decreased CASTOR1 protein level and activated mTORC1 with and without the presence of arginine. c Overexpression of a myristoylated constitutively active AKT1 (myr) but not the kinase-dead AKT1 mutant (K179M) reduced CASTOR1 protein level, decreased its binding to MIOS, and activated mTORC1. d AKT regulated mTORC1 activity by suppressing CASTOR1 was independent of TSC2. e–g CASTOR1 S14D had a weaker binding to MIOS shown by CASTOR1 co-immunoprecipitation (co-IP) of MIOS (e) and reversed MIOS co-IP of CASTOR1 (g), hence S14D had a less inhibitory effect on mTORC1 than WT and S14A had (e, g), and quantification of results from two independent experiments shown in f. h An illustration depicting that AKT phosphorylation and RNF167 ubiquitination of CASTOR1 reverse CASTOR1 inactivation of mTORC1. Blots in a–d and g are representatives of n = 3 independent experiments, and blots in e are representatives of n = 2 independent experiments. Source data are provided in Source data file.

Fig 4: AKT1 phosphorylation of CASTOR1 promotes RNF167-mediated ubiquitination and degradation of CASTOR1.a Deprivation of arginine or leucine activated AKT and increased CASTOR1 phosphorylation at S14, whereas deprivation of FBS or total amino acids inactivated AKT and reduced CASTOR1 phosphorylation at S14. b CASTOR1 S14 phosphorylation was markedly reduced following alanine substitution (S14A). c AKT inhibition abolished CASTOR1 phosphorylation at S14 in vivo. d Recombinant AKT1 protein directly phosphorylated CASTOR1 protein in vitro. e, f AKT overexpression increased (e) while AKT1 knockdown decreased (f) CASTOR1 degradation. g, h AKT1 overexpression increased (g) and AKT1 knockdown decreased (h) CASTOR1 ubiquitination. i CASTOR1 S14D had increased ubiquitination level compared to WT and S14A. j, k Phosphorylation of CASTOR1 at S14 significantly increased its affinity to RNF167 (j), and quantifications of results from three independent experiments are presented (k). For k, data are presented as mean values ± SEM and P value was calculated by one-way ANOVA followed by Tukey post hoc test (n = 3 independent experiments). Blots in a–c, e–j are representatives of n = 3 independent experiments, and blots in d are representatives of n = 2 independent experiments. Source data are provided in Source data file.