Molecular mechanisms of KRas4b signaling

Shree, Shweta

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https://hdl.handle.net/2142/129718 Copy

Description

Title

Molecular mechanisms of KRas4b signaling

Author(s)

Shree, Shweta

Issue Date

2025-05-02

Director of Research (if dissertation) or Advisor (if thesis)

Sligar, Stephen G.

Doctoral Committee Chair(s)

Sligar, Stephen G.

Committee Member(s)

• Chen, Jie
• Zhang, Kai
• Stephen, Andrew G.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Cancer signaling
• KRas4b
• Lipid specificity
• Membrane topology
• Nanodisc
• KRas4b trafficking
• Calmodulin
• Electrostatics
• Raf-1.

Abstract

Ras (Rat Sarcoma Virus) proteins are expressed in all animal cells and organs and are essential components of signalling pathways originating from cell surface receptors. Oncogenic mutations that lock Ras in its active, GTP-bound state are responsible for nearly 85% of Ras-driven cancers, including pancreatic, colorectal, and lung tumors. KRas4b is a membrane-associated regulatory protein from the GTPase family that functions as a GTP/GDP switch, mediating signal transduction from activated membrane receptors to intracellular targets. Its interactions with effector molecules predominantly occur at the inner leaflet of the plasma membrane. These protein-protein interactions are influenced by the KRas4b "hypervariable" region (HVR) and the "globular" domain, which interact through anionic lipids and a farnesyl anchor embedded in the bilayer. Active, membrane-bound KRas4b facilitates the recruitment of its downstream effector, Raf-1. Raf-1 contains an N-terminal regulatory domain and a C-terminal kinase domain. The N-terminal region comprises the Ras-binding domain (RBD) and the cysteine-rich domain (CRD), which engages with phospholipids. The assembly of the KRas4b Raf-1 complex on the membrane is believed to be driven by cooperative interactions among membrane lipids, KRas4b, and Raf-1. However, the precise mechanism and spatial arrangement of the Ras/RBD/CRD ternary complex on the membrane remain unclear. The topology of the KRas4b G-domain relative to the membrane surface, the influence of lipid head groups on its positioning, and how these factors regulate effector interactions and signaling outcomes are not fully understood. KRas4b is trafficked to and from the plasma membrane to modulate the capacity and frequency of Ras Signaling. The interaction of chaperones and KRas4b on the membrane surface, and the role of lipids facilitating this trafficking mechanism is not fully understood. In my doctoral research, I developed a fluorescence resonance energy transfer (FRET) system to elucidate the topology of fully post-translationally modified KRas4b-FME on Nanodisc membranes. Using this Nanodisc-based FRET assay, I investigated how lipid composition affects the topology of KRas4b-FME and its interactions with Raf-1 RBD/CRD. By measuring FRET donor-acceptor distances as a function of bilayer charge, I found that a negatively charged membrane surface weakly promotes closer association with the bilayer. Extending these measurements to complexes with Raf-1 shows that the ternary KRas4b- Raf-1 RBD/CRD favors a closer proximity to the lipid bilayer surface, and anionic lipids reorients the ensemble average closer to the membrane surface. These measurements supports the ‘fly casting’ model for recruitment and formation of active signaling complexes. In my experiments, I utilize Biolayer Interferometry and Homogeneous Time Resolved Fluorescence to study the role of KRas4b in enhancing the recruitment of Raf-1 RBD/CRD to the lipid bilayer. Using Biolayer interferometry, I also assayed the role of lipid head group, chain length, and electrostatics in the dissociation kinetics of KRas4b from Nanodisc bilayers with defined lipid compositions. My results suggest that Calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS-containing bilayers. In addition to this, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers showing that the overall charge of the bilayer, the identity of the head groups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.

Graduation Semester

2025-05

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129718

Copyright and License Information

Copyright 2025 Shweta Shree

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