LAUSR

The Hippo pathway utilizes a well-characterized Ser/Thr kinase cascade to control the downstream effectors, Yap and Taz. In addition, Yap/Taz and other Hippo pathway components are directly regulated by tyrosine kinases (TKs). The methodological strategies described here use the example of the c-Abl non-receptor TK and the Yap substrate to outline the steps used to identify and to validate tyrosine phosphorylation sites, including bioinformatic approaches, ectopic expression of proteins in transfected tissue culture cells, and mutagenesis of endogenous proteins by CRISPR-Cas9. These general strategies can be applied to investigate regulation of protein signaling moieties by tyrosine phosphorylation in the context of distinct TKs.