Fig. 5: WNK1-OXSR1/STK39 pathway controls mTORC1 signalling.

a Experimental workflow for the phosphoproteome analysis. b Volcano plot showing changes in the phosphoproteome in MA9 leukaemia cells treated with 30 µM Compound 12 for 3 h. Significantly regulated phosphosites of mTORC1 downstream targets are highlighted in colour (FDR  <  0.05). Statistical analysis was performed using the MSstatsTMT package with two-sided tests and Benjamini-Hochberg correction for multiple comparisons. Adjusted p-values (FDR < 0.05) were used to determine significance. c Schematic depicting WNK1 depletion/inhibition leads to inhibition of mTORC1 signalling. d Immunoblot analysis of the indicated mTORC1 downstream proteins of Wnk1^fl/− MA9 leukaemia cells in the presence of OHT (500 nM, 48 h), Compound 12 (C12) (10 µM, 1 h), WNK463(W463) (10 µM, 1 h), Rapamycin (RAPA) (10 µM, 1 h). Blots are representative of at least three independent experiments (n > 3). The samples were derived from the same experiment, but different gels for p-OXSR1(S325) and p-4EBP1(T37/46), another for p-P70S6K (T389) and p-4EBP1/2/3(T45), another for Actin and 4EBP1 (Total), and another for 4EBP2 (Total) were processed in parallel. Ponceau S staining and Actin served as loading controls. e Immunoblot analysis of the indicated mTORC1 downstream proteins of Wnk1^fl/− MA9 leukaemia cells ectopically expressing OXSR^WT, STK39^WT, OXSR1^{T185E, S325E} or STK39^{T233E, S373E}. The cells were treated with OHT (500 nM) for 48 h. Blots are representative of two independent experiments (n = 2). The samples were derived from the same experiment, but different gels for OXSR1 (Total) and p-4EBP1(T37/46), another for STK39 (Total) and 4EBP1 (Total), another for p-P70S6K (T389) and p-4EBP1/2/3(T45), another for P70S6K (Total) and 4EBP2 (Total), and another for Actin were processed in parallel. Actin served as a loading control. f Left, schematic of the degron system for the targeted degradation of WNK1 in MA9 leukaemia cells. Right, immunoblot analysis of indicated proteins at the indicated times after treatment with 500 nM dTAG-13. Blots are representative of three independent WNK1-degron clones (n = 3). The samples were derived from the same experiment, but different gels for WNK1 and p-OXSR1(S325), another for HA and p-P70S6K (T389), another for P70S6K (Total) and Vinculin were processed in parallel. Ponceau S staining and Vinculin served as loading controls. g Protein synthesis rates as measured by incorporation of OP-puro in Wnk1^fl/− MA9 leukaemia cells treated with 10 µM Compound 12 at the indicated time points. Error bars represent mean ± SD from three biological replicates (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test to compare each Compound 12-treated time point to the untreated control.