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

ABSTRACT: 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 causes cell competition-mediated extrusion and apoptosis of only acinar progenitors. Csk mutants can be phenocopied by constitutively active Yap and rescued by the genetic ablation of Yap or Taz, indicating remarkable functional equivalence between Src and Yap signaling. Although Csk inactivation induces tyrosine phosphorylation of Yap, mutating these tyrosine residues in both Yap and Taz fails to alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Chemical genetics studies further demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas the deletion of either Crk/CrkL or Rac1/Rap1 rescues the Csk mutant phenotype. These results show that Csk controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to regulate cell competition.

ORGANISM(S): Mus musculus

PROVIDER: GSE253784 | GEO | 2025/01/19

REPOSITORIES: GEO

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Project description:The SRC inhibitor PP2 can block CASK nucleus translocation in macrophage under H5N1 infection. Since PP2 is a general inhibitor for Src family kinases (SFKs), which includes nine members (Csk, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk), we aimed to identify the specific member responsible for the nuclear translocation of CASK. First, we examined the expression of SFKs in macrophages (Csk, Fyn, Fgr, and Hck) following H5N1-IAV infection (Fig. 4A). This observation suggests that Hck is the most abundant SFK and may contribute to virus-induced nuclear translocation of CASK. To test this, we generated constitutively active forms of HCK: HCK△513-524 (deletion mutant) and HCK Y499F mutant (YF mutation) [24], then co-transfected them with an EGFP-CASK construct into 293T cells. Immunoprecipitation with anti-GFP mAb, followed by trypsin digestion and LC-MS/MS analysis, revealed that compared to the control group, the phospho/nonphospho ratio at residue Serine 395 (S395) of CASK increased approximately 10-fold (0.008 to 0.072 by HCK△513-524, 0.008 to 0.108 by HCK Y499F), while the other 8 potential phosphorylation sites did not show significant alterations (Table 1). To confirm the phosphorylation of S395 of CASK is required for nuclear translocation, we generated an EGFP-CASK S395A mutant (SA mutation) to prevent CASK phosphorylation. In the absence of HCK Y499F, both the wild type (CASK S395) and mutant (CASK S395A) remained in the cytosol (left two columns, Fig. 4B). In contrast, transfection of HCK Y499F (red color) induced the translocation of wild type CASK (green color) into the nucleus (3rd column from left, Fig. 4B), an effect not observed with the CASK S395A mutant (4th column from left, Fig. 4B). The percentage of nuclear GFP-CASK (60% and 90% in WT and 35% in S395A) resulting from the overexpression of wild type HCK and HCK Y499F is shown in Figure 4C. Thus, we concluded that activated HCK induces the phosphorylation of CASK at Ser395, facilitating its nuclear translocation.

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

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