Proteomics- and BRET- screens identify SPRY2 as a Ras effector that impacts its membrane organization.

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Regulators of Kirsten rat sarcoma viral oncogene homolog (K-Ras) plasma membrane organization are largely unknown.
• Understanding K-Ras regulation is crucial for cancer research and targeted therapies.

Purpose of the Study:

• To identify novel regulators of K-Ras G-domain organization at the plasma membrane.
• To investigate the role of identified interactors, particularly SPRY2, in K-Ras signaling and cellular processes.

Main Methods:

• TurboID-based proximity proteomics to identify K-Ras interactors.
• Bioluminescence Resonance Energy Transfer (BRET) assays to study protein-protein interactions.
• Co-immunoprecipitation and mutation analysis to validate interactions and binding interfaces.
• Cellular differentiation assays (C2C12 muscle cells) to assess functional impact.

Main Results:

• Eight potential K-Ras G-domain interactors were identified, including APLP2 and SPRY2.
• SPRY2, particularly its C-terminal half, binds oncogenic RasG12V at the plasma membrane, dependent on K-Ras activity and membrane anchorage.
• SPRY2 and its fragment promote C2C12 muscle cell differentiation, a process linked to MAPK pathway inhibition.
• SPRY2 forms homo- and hetero-oligomers with SPRY4.

Conclusions:

• Active K-Ras recruits SPRY2 dimers to the plasma membrane.
• SPRY2 acts as a K-Ras effector regulator, potentially by blocking Ras effector access.
• SPRY2 plays a role in cellular differentiation processes requiring MAPK pathway modulation.