Two-Step Mechanism of Bruton's Tyrosine Kinase Membrane Recruitment and Activation

Peripheral membrane proteins (PMPs) are critical mediators of signaling cascades initiated via activation of cell-surface receptors. Their functions rely on their innate ability to interact with membranes dynamically in response to rapidly changing cellular conditions. This membrane recruitment may occur via high-affinity interactions with specific lipids or transient, low-affinity membrane interactions. These weak and dynamic interactions that are critical regulators of protein function are challenging to capture. Taking Bruton's Tyrosine Kinase (BTK), a non-receptor tyrosine kinase essential for B cell activation, as an example, we demonstrate a native mass spectrometry (nMS) platform to understand the recruitment of PMPs by directly studying it from lipid bilayers customized to target membranes. Our data demonstrates that BTK recognizes phosphatidylserine (PS) through sites distinct from phosphatidylinositol (3,4,5) phosphate (PIP₃) binding. We show PS-bound BTK retains PIP₃ binding simultaneously via high-affinity sites, exhibiting PIP₃-independent basal membrane recruitment of BTK. Biochemical assays show that this PS-mediated recruitment sensitizes BTK to PIP₃-mediated activation under near-physiological concentrations of PIP₃. Thus, we propose a two-step model for BTK membrane recruitment and activation. A low-affinity interaction with high-copy number PS enables plasma membrane recruitment of BTK and increases its membrane-bound concentration. Upon B-cell activation, this pre-recruited, membrane-bound BTK population localizes to PIP₃-rich domains through electrostatic gliding along the membrane driven by low-affinity PS and high-affinity PIP₃ binding. This indicates a cooperative mechanism where PS can amplify B-cell signaling through increased membrane-bound BTK concentration. Our work demonstrates a general model of PH-domain-containing protein regulation by weak protein-lipid interactions, which can be extended to many other PMPs.