Exploring the phosphor-proteome in B16 cells wild type cells or TAK1 CRISPR KO cells upon exposure to combined TNF and Interferon Gamma stimulation.

Global phosphoproteomic profiling of wild-type and TAK1-deficient B16F10 cells treated with TNF (10ng/ml) and IFNγ (1ng/ml) for one hour. The analysis reveals that TAK1 loss selectively disrupts phosphorylation of NF-κB and MAPK pathway components, including RELA, RIPK2, and MAP3K8, while STAT1-associated phosphorylation events remain intact, indicating that IFNγ–STAT1 signaling is TAK1 independent. Stratification of phosphosites highlights TAK1-sensitive sites, which are induced in wild-type but lost in TAK1 knockout cells, and TAK1-independent sites, which remain unaffected by TAK1 loss. Notably, phosphorylation of RIPK1 was altered: the inhibitory S25 site was reduced in TAK1-deficient cells, while a cluster of sites spanning Y309–S415 was more prominently phosphorylated, consistent with enhanced pro-death signaling. Together, these data show that TAK1 sustains NF-κB–driven survival signaling while restraining RIPK1 activation, thereby acting as a molecular switch that biases TNF and IFNγ signaling toward cytoprotection; its loss rewires cytokine signaling toward apoptosis.