Identification of protein interactors of BRAF via Chemical Induced Dimerization‐BRAF and LC‐MS/MS

Abstract

Rapidly Accelerated Fibrosarcoma (ARAF, BRAF, CRAF) Kinase is central to the MAPK Signaling Cascade (RAS‐RAF‐MEK‐ERK). Despite over three decades of scrutiny, the regulatory mechanisms of BRAF are not well understood. According to the accepted model, inactive RAF kinase is monomeric, autoinhibited, and cytosolic while activated RAF is membrane recruited via RAS‐GTP and dimerized; however—direct evidence and key details are still missing. Using a chemical biology approach to target this complex biological system, we seek to exert spatial control using the genetically encodable Chemically Induced Dimerization (CID)‐BRAF; CID‐BRAF exploits the FRB‐rapamycin‐FKBP system to membrane recruit BRAF. We hypothesize that membrane recruitment of BRAF enables diversification of PPI, which remain elusive due to the transient nature of these interactions. To capture transient protein‐protein interactions (PPI), cytosolic BRAF and membrane‐recruited BRAF cells were crosslinked, co‐immunoprecipitated, and identified via LC‐MS/MS. Current efforts are focused on the validation of putative MS identified PPI and identification of BRAF interaction sites. Identifying novel PPI will provide additional mechanistic insight to the regulation of BRAF and facilitate the development of next generation inhibitors of dysregulated BRAF.