Youngkwang Lee, Ph.D.
Abstract
Raf is an important kinase that initiates the three-tiered Raf-MEK-ERK signaling cascade in response to diverse receptor stimuli. Understanding the regulation of Raf membrane binding is crucial to fully grasp its activation mechanisms, as Raf activation exclusively occurs on membrane surfaces. However, there is a notable lack of quantitative experimental studies exploring how Raf integrates both protein and lipid inputs for proper activation and deactivation. To address this gap, we used single-molecule total internal reflection fluorescence microscopy (smTIRF) and fluorescence correlation spectroscopy (FCS) to examine the molecular binding and diffusion of Raf on supported lipid membranes. Our findings reveal functional coupling between the Ras binding domain (RBD) and the cysteine-rich domain (CRD) within Raf’s regulatory region. This coupling gives rise to distinct modulation of the membrane association and dissociation rates of Raf, demonstrating a strong dependency of membrane binding kinetics on the surface density of Ras and anionic lipids. Additionally, we investigated how Ras-Raf complex formation and membrane environments resist deactivation by GTPase-activating proteins (GAPs). Our results provide new insights into the kinetic mechanisms underlying Raf activation, particularly the release of its autoinhibited complex and the transition from monomer to dimer in the context of Ras nanoclusters.
When: November 22, 2024
Where: North Classroom 1130
Time: 11:00 am - 12:00pm