Because CRAF is the key isoform of RAF kinase responsible for the RAF inhibitor-induced paradoxical activation of RAF–MEK–ERK signaling in cancer therapy, in this study, we used it as a module to explore the molecular mechanism that governs 14-3-3 binding to the C terminus of RAF kinase and thus regulates its dimerization-driven transactivation. This evidence concerns the gene MAPK1 and cancer.