Isobel Barlow-Busch

Topic

Molecular mechanisms involved in regulating the lipid kinases PIKfyve and phosphoinositide 3-kinase alpha (PI3Kα)

School of Molecular Life Sciences

Date & location

• Tuesday, October 20, 2026
• 10:00 A.M.
• Clearihue Building, Room B007

Examining Committee

Supervisory Committee

• Dr. John Burke, School of Molecular Life Sciences, University of Victoria (Supervisor)
• Dr. Alisdair Boraston, School of Molecular Life Sciences, UVic (Member)
• Dr. Jennifer Cobb, School of Molecular Life Sciences, UVic (Member)
• Dr. Leigh Anne Swayne, School of Medical Sciences, UVic (Outside Member)

External Examiner

• Dr. Lois Weisman, Department of Cell and Developmental Biology, University of Michigan

Chair of Oral Examination

• Dr. Adam Krawitz, Department of Psychology, UVic

Abstract

Phosphoinositide kinases regulate a wide range of cellular processes by generating phosphoinositide signalling lipids that control membrane dynamics, intracellular trafficking, growth, and metabolism. This dissertation investigates the regulation of two phosphoinositide kinases with important roles in human disease: PIKfyve and phosphoinositide 3-kinase α (PI3Kα). The first focus of this thesis, PIKfyve, generates PI(3,5)P₂ on endolysosomal membranes, where it is essential for lysosomal homeostasis and membrane trafficking. Although PIKfyve has emerged as a promising therapeutic target for neurodegenerative disease, viral infection, and cancer, the function and regulation of this enzyme remain incompletely understood. The second focus of this thesis, PI3Kα, generates PI(3,4,5)P₃ at the plasma membrane to regulate cell growth, proliferation, survival, and metabolism. PI3Kα is among the most frequently dysregulated signalling enzymes in human cancer and is the focus of ongoing efforts to develop mutant-selective therapeutics.

To investigate the molecular mechanisms governing the regulation of these enzymes, this dissertation combines biochemical, biophysical, and structural approaches, including kinase activity assays, protein-lipid fluorescence resonance energy transfer (FRET), and hydrogendeuterium exchange mass spectrometry (HDX-MS). First, I characterized a minimal, catalytically active fragment of human PIKfyve and a PIKfyve-selective nanobody that provides a platform for future structural and mechanistic studies. Second, I examined how the five class IA PI3K regulatory subunit isoforms influence PI3Kα activity and demonstrated that they regulate the p110α catalytic subunit through indistinguishable biochemical mechanisms in vitro. Finally, I characterized a panel of oncogenic PIK3R1 mutations identified in breast cancer patients, demonstrating that these variants relieve autoinhibition, promote membrane recruitment and kinase activation, and remain sensitive to the next-generation allosteric inhibitor STX-478.

Collectively, this work provides new mechanistic insights into the regulation of PIKfyve and PI3Kα under physiological and disease-associated conditions. These findings establish new experimental tools for investigating PIKfyve, clarify fundamental aspects of PI3Kα regulation, and identify therapeutic vulnerabilities associated with oncogenic PIK3R1 mutations, advancing our understanding of lipid kinase biology and informing the development of targeted therapies.