Conditional Requirement for Dimerization of the Membrane-Binding Module of BTK

Abstract

Bruton’s tyrosine kinase (BTK) functions in many types of immune cells and is a major chemotherapeutic target. BTK is activated at the plasma membrane and can dimerize through its pleckstrin-homology and contiguous tec-homology domains (the PH–TH module). The features that support dimerization of the PH–TH module are unique to BTK among the Tec kinases and appeared relatively recently in evolutionary history. Previous studies suggest that dimerization enhances kinase activity by promotingtransautophosphorylation, but whether BTK dimerizes in cells via the PH–TH module, and whether this dimerization controls signaling, is unknown. To address this question, we developed a high-throughput mutagenesis assay for BTK function in B cells and T cells. We measured the fitness costs for thousands of point mutations in the PH–TH module and kinase domain, allowing us to assess whether dimerization of the PH–TH module and BTK kinase activity are necessary for function. In Ramos B cells we find that neither dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations that increase membrane adsorption, even at the cost of reduced PH–TH dimerization. In contrast, in Jurkat T cells, we find that BTK signaling depends on PH–TH dimerization and also on kinase activity. These results provide a framework for understanding why PH–TH dimerization is a more recent evolutionary innovation that is not conserved across the Tec family.