Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion.

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. mutants can be phenocopied by constitutively active and rescued by deleting or , indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both and fails to perturb mouse development or alleviate the lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing / or / ameliorates the mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.