Proteomics- and BRET-screens identify SPRY2 as Ras effector that impacts its membrane organisation

K-Ras functions within nanoscale proteo-lipid domains of the plasma membrane, but few regulators of its membrane organisation are known. We combined TurboID-based proximity proteomics with a secondary BRET screen to identify eight novel K-Ras G-domain interactors. We focused on APLP2 and SPRY2 for further characterisation. APLP2 binds K-Ras indirectly via C-Raf, while SPRY2 exhibits properties of a novel effector. Co-immunoprecipitation and BRET assays revealed that the SPRY2 C-terminal fragment (residues 161–315) binds oncogenic RasG12V more strongly than the full-length protein. Both forms localise to the plasma membrane, but this localisation and binding to K-Ras is disrupted by inhibitors of K-Ras membrane anchorage or activity. Mutations at the predicted interface of K-Ras and SPRY2’s C-terminal region affect the interaction. Both full-length SPRY2 and its C-terminal fragment promote differentiation of C2C12 muscle cells, a process requiring MAPK pathway inhibition. Finally, SPRY2 also forms homo- or hetero-oligomers with SPRY4. We propose that active K-Ras recruits SPRY2 dimers to the membrane, where they bind Ras and block effector access.