In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function

Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival and inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has for more than two decades generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using non-lymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas mediated knock-in mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness, while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and morphology of lymphoid organs as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control, and mounted similar humoral immune responses as Y223F and wildtype animals. Collectively our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase C2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this post-translational modification is dispensable for the function of BTK.