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

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Bruton's tyrosine kinase (BTK) is crucial for B-cell survival and a target for B-cell malignancy treatments.
• Phosphorylation of tyrosine 551 in BTK's catalytic domain is an initial activation event.
• Tyrosine 223 (Y223) in the SRC homology 3 (SH3) domain has been considered vital for BTK's enzymatic activity for over two decades.

Purpose of the Study:

• To investigate the biological significance of Y223 phosphorylation in BTK's function.
• To determine if Y223 phosphorylation is essential for B-cell survival and immune responses.

Main Methods:

• Generated CRISPR-Cas-mediated knockin mice with a Y223F substitution to prevent Y223 phosphorylation.
• Assessed leukocyte subsets, lymphoid organ morphology, and humoral immune responses in wild-type and Y223F mutant mice.
• Utilized C481S mutant mice, resistant to BTK inhibitors, as an additional control.

Main Results:

• No detectable differences were observed in leukocyte subsets, lymphoid organ morphology, or humoral immune responses between wild-type and Y223F mutant mice.
• Humoral immune responses in Y223F and C481S mutant mice were comparable to wild-type animals.
• Phosphorylation of Y223 is dispensable for BTK function, contrary to previous beliefs.

Conclusions:

• Y223 phosphorylation is not required for BTK's enzymatic activity or its role in B-cell function.
• Y223 phosphorylation may serve as a proxy for phospholipase Cγ2 (PLCG2) phosphorylation, BTK's endogenous substrate.
• This study revises the understanding of BTK regulation and its functional requirements in the immune system.