Ghaffari-Kashani, S. | bioRxiv Science

bioRxiv2025

BTK Autoinhibition Analyzed by High-Throughput SH2 Domain Swaps

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, positioning the SH3 domain in an inhibitory conformation; in Abl, interaction between the kinase domain and a myristoyl group on the N- terminal segment provides the same latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an SH2-kinase latch. To assess autoinhibition in BTK, we generated hundreds of chimeric BTK molecules and measured their fitness using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins demonstrated that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. These results reveal the importance of distributed interactions between the SH2 and kinase domains of BTK in stabilizing the inhibitory conformation, and suggest that the specialized latching mechanisms in Src and Abl kinases may be later evolutionary refinements.

biochemistry↗

bioRxiv2023

Differential Requirement for Dimerization of the Membrane-Binding PH-TH Module of BTK in B cells and T cells

Brutons tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin-homology and tec-homology (PH-TH) domains of BTK are required for signaling. In vitro, dimerization of the PH-TH module strongly stimulates BTK kinase activity. Whether BTK dimerizes in cells via the PH-TH module, and whether this dimerization is necessary for signaling, is unknown. To address this question, we developed high-throughput mutagenesis assays 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 PH-TH dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations in the PH-TH module 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 both PH-TH dimerization and kinase activity. Evolutionary analysis shows that BTK proteins in fish and lower organisms, like all Tec kinases other than BTK, lack PH-TH dimerization but have active kinase domains. Thus, PH-TH dimerization is not intrinsically required for Tec-kinase activity, and is a special feature that evolved to exert stricter regulatory control on BTK kinase activity as adaptive immune systems gained increased complexity.

biochemistry↗