Paradoxical non-catalytic kinase functions are driven by inhibitor-induced displacement of autoinhibitory domains.
ATP-competitive kinase inhibitors represent one of the largest classes of targeted anti-cancer drugs. While their primary mechanism is to block catalytic activity, they can also trigger paradoxical phenotypic effects that cannot be explained by catalytic inhibition alone. These observations point to a hidden layer of drug action that modulates non-catalytic kinase functions via changes in kinase conformation and protein-protein interactions (PPIs). Here, we developed a multimodal proteomics approach combining limited proteolysis coupled mass spectrometry on affinity-purified samples (AP-LiP-MS), AP-MS, and proximity labeling-MS to map inhibitor-induced conformation and PPI changes. We show that inhibitor binding causes structural rearrangements in the autoinhibitory domains (AIDs) of all tested kinases, consistent with a transition to an open, active-like kinase conformation. These structural shifts drive distinct kinase-protein interaction changes that control non-catalytic functions: sequestration of AMPK by inhibited CAMKK2 blocks phosphorylation by other kinases, CHEK1 inhibition causes dissociation from the mitochondrial protein CLPB and leads to mitochondrial fragmentation, and structural changes in inhibited PRKCA trigger rapid relocalization to cell junctions. Thus, we identify the ATP-binding site as a major organizing center of kinase conformation and interaction. Our work suggests that these on-target, off-mechanism effects are likely to occur in other kinases as well, and provides the analytical framework to systematically characterize a frequently overlooked phenomenon highly relevant for understanding drug side effects to guide the development of novel therapeutics.