Journal: bioRxiv
Article Title: Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion
doi: 10.1101/2024.06.27.601065
Figure Lengend Snippet: (A) Cre-mediated depletion of Csk in MEF cells led to the loss of pSrc Y527 inhibitory phosphorylation and a concomitant increase in pSrc Y416 activating phosphorylation without perturbing ERK phosphorylation. (B) CRISPR-Cas9 genome editing was used to mutate two tyrosine phosphorylation sites ( Yap Y341F/Y357F or Yap Y2F ). (C) Luminal dilation and Mist1 expression defect were not rescued in Csk CKO ; Yap Y2F mutants. (D) Yap tyrosine phosphorylation was reduced but not lost in Yap Y2F homozygous MEF cells. (E) Mutating all three tyrosine residues in a Flag-tagged Yap (Yap Y3F ) led to a complete loss of tyrosine phosphorylation as revealed by Flag or phospho-tyrosine (pY) immunoprecipitation. (F) Schematic diagram of CRISPR-Cas9 genome editing to generate Yap Y341F/Y357F/Y394F ( Yap Y3F ) and Taz Y321F ( Taz YF ) mutant mice. (G) Yap protein remained nuclear in Csk CKO ; Yap Y3F ; Taz YF mutants, resulting in luminal dilation and loss of Mist1 expression. (H) The lumen size was quantified as the ratio of the lumen area versus the overall epithelial area. One-way ANOVA, n=5, *P<0.01. (I) Quantification of Mist1+ cells. One-way ANOVA, n=5, *P<0.0001. Scale bars: 100 µm.
Article Snippet: Plasmids encoding Yap 2YF and Yap 3YF were constructed from pcDNA Flag-Yap1 Y357F (Addgene #18882) through site-directed mutagenesis using Neb Q5 high-fidelity polymerase (New England Biolab #M0491).
Techniques: Phospho-proteomics, CRISPR, Expressing, Immunoprecipitation, Mutagenesis
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