Molecular mechanism of membrane association of Bruton's Tyrosine Kinase

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PROJECT SUMMARY The adaptive immune system is driven by the B Cell receptor (BCR) pathway, which triggers B cell differentiation and proliferation in response to antigen binding. Activation of the BCR generates signaling lipid phosphatidylinositol – 3,4,5 – triphosphate (PIP3) on the inner leaflet of the plasma membrane. The pleckstrin homology (PH) domain of non-receptor tyrosine kinase Bruton's Tyrosine Kinase (Btk) binds to this PIP3, triggering release of an auto-inhibited conformation and trans auto-phosphorylation. Auto- phosphorylated Btk activates downstream pathways, leading to B cell activation and proliferation. Overactive BCR signaling can lead to severe malignancies, such as chronic lymphatic leukemia and non-Hodgkin's lymphoma. The first Btk inhibitor, ibrutinib, was approved in 2013 by the Food and Drug Administration as an alternative to chemotherapy for the treatment of B cell malignancies. Successful Btk inhibition slows cancer cell proliferation by reducing the activation and binding of transcription regulator NF-κB to DNA. The most widely used Btk inhibitors are ibrutinib and second-generation derivatives, which irreversibly bind the ATP-binding pocket of the kinase domain. This pocket is highly conserved among tyrosine kinases and consequently, treatment leads to significant off-target side effects and resistance due to mutations in the binding site. These factors necessitate alternative inhibitory sites within Btk for the advancement of B-cell cancer treatment. The critical and initial step in Btk activation is its plasma membrane association through the PH domain. The PH domain represents an attractive inhibitory target as there is low sequence homology among the class. However, the lipid specificity, stoichiometry of PIP3 binding and how it regulates these assemblies, functional oligomeric states of full-length Btk, the interfaces involved, are unknown. The goal of my proposal is to determine the lipid specificity, stoichiometry, mechanism of membrane recruitment, and the membrane-associated oligomeric states of Btk. Through a quantitative understanding of these molecular events, I aim to understand how the function of Btk at the plasma membrane is regulated. I will use a workflow developed by the Gupta lab to directly detect protein-protein and protein-lipid interactions from a lipid bilayer using native mass spectrometry (nativeMS). This will allow me to detect the lipids that interact with Btk in a bilayer mimicking the lipid composition of the plasma membrane as well as the oligomeric states of membrane bound Btk. I have obtained preliminary nativeMS and vesicle association data that shows in vitro binding to PS as well as the ability to associate with bilayers in a PS-dependent manner. I will determine the functional consequences of this yet uncharacterized lipid interaction using an immortalized B Cell line. By activating the BCR within the presence of a PS scavenger, I can determine whether PS plays a role in the BCR pathway, making it a mechanism for inhibition of Btk.