# Molecular mechanism of membrane association of Bruton's Tyrosine Kinase

> **NIH NIH F31** · YALE UNIVERSITY · 2023 · $45,764

## Abstract

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 BC...

## Key facts

- **NIH application ID:** 10604872
- **Project number:** 1F31CA278383-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Rachel McAllister
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $45,764
- **Award type:** 1
- **Project period:** 2023-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10604872

## Citation

> US National Institutes of Health, RePORTER application 10604872, Molecular mechanism of membrane association of Bruton's Tyrosine Kinase (1F31CA278383-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10604872. Licensed CC0.

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