Fig. 4: ZAKα activates the SRC-YAP axis during ribosome impairment and results in ISCs identity switch in vivo during leucine deprivation.

A Volcano plot depicting differentially enriched interactors of ZAKα in Rptor^fl/fl organoids, using RIME-MS (n = 2 biological replicates). An empty vector tagged FLAG bait is used as a control. Significance when p-value ≼ 0,05 (t-test, two-tailed). B Immunoprecipitation of ZAKα-FLAG confirms interaction with SRC in organoids treated with cycloheximide (0.015 ug/ml) and deprived from glutamine. EV_FLAG is used as a control. One biological animal was used. C Quantification of the western blot shows enrichment of ZAKα-SRC binding in organoids treated with cycloheximide (0.015 ug/ml) and deprived from glutamine, compared with wild type cells. D Western blot showing activation of SRC in Rptor^fl/fl, low dose cycloheximide and glutamine-deprived organoids compared to wild type. This activation is dependent on ZAKα activity, as inhibition with vemurafenib abolishes SRC phosphorylation (T416). β-actin serves as a loading control. Blue panel has a higher exposure. Blots were done on one animal. E Western blot showing activation of YAP in Rptor^fl/fl, low dose cycloheximide and glutamine-deprived organoids compared to wild type. This activation is dependent on SRC, as inhibition with dasatinib abolishes YAP phosphorylation (Y357). β-actin serves as a loading control. Blue panel has a higher exposure. Blots were done on one animal. F Gene Set Enrichment Analysis based on RNASeq differential expression data comparing Rptor^fl/fl organoids to WT (n = 4 from 2 biological replicates for each). Enrichment is shown for transcriptional signatures related to YAP-target genes. p-values were determined using the clusterProfiler package. G RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT and Rptor^fl/florganoids treated with inhibitors for SRC (dasatinib, 100 nM, 24 h) and YAP (verteporfin, 3 uM for 24 h). Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test. H Experimental workflow of the dietary interventions performed in WT and ZAK KO mice. I RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of colons of WT and ZAK KO mice. Hprt is used as a reference. Mean and SEM are shown (n = 5 (WT) and 9 (ZAK KO) biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test.