Proteomics- and BRET-screens identify SPRY2 as interactor of active Ras the impacts on its membrane organisation

ABSTRACT: K-Ras is regulated and active in nanoscale proteo-lipid domains of the plasma membrane. Few regulators of K-Ras membrane organisation have been identified so far. We here combined a primary TurboID-based proximal proteome screen with a secondary BRET-screen to identify nine novel bona fide interactors of the K-Ras G-domain that would modulate its membrane organisation. We focused our hit characterisation on one novel candidate, APLP2, and SPRY2, which was previously implicated as negative regulator of MAPK-signalling. While APLP2 appeared to indirectly bind to K-Ras via C-Raf, SPRY2 showed characteristics of a new effector protein. We show by co-immunoprecipitation and BRET-experiments that the C-terminal fragment of SPRY2 comprising residues 161-315 interacts more with K-RasG12V than the full-length protein. Both full length and C-terminal fragment localized to the membrane. SPRY2 plasma membrane localization was blocked if K-Ras membrane anchorage was inhibited. Likewise, binding to oncogenic K-Ras was disrupted by K-Ras-inhibitors. Mutations at the predicted interface of the K-Ras effector binding region and the C-terminal fragment of SPRY2 modulated the interaction. Our data further suggest that SPRY2 operates as homo- or hetero-di/oligomer with SPRY4. Both full length SPRY2 and its C-terminal fragment promote differentiation of muscle C2C12 cells, which requires inhibition of MAPK-signalling. We propose a model, wherein active K-Ras recruits SPRY-dimers via the C-terminus of SPRY2 to the plasma membrane where they bind directly to Ras and block access of effectors.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Fiona Hood  

LAB HEAD: Ian A Prior

PROVIDER: PXD067415 | Pride | 2025-10-02

REPOSITORIES: Pride